Monoclonal Antibodies That Inhibit The Wnt Signaling Pathway and Methods of Production and Use Thereof

ABSTRACT

Monoclonal antibodies against LRP6 and that block the Wnt signaling pathway are disclosed. Methods of production and use thereof are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS/INCORPORATION BY REFERENCESTATEMENT

The present application is a continuation of U.S. Ser. No. 14/299,570,filed Jun. 9, 2014; which is a divisional of U.S. Ser. No. 13/031,010,filed Feb. 18, 2011, now U.S. Pat. No. 8,859,736, issued Oct. 14, 2014;which claims benefit under 35 U.S.C. 119(e) of U.S. provisionalapplication Ser. No. 61/306,083, filed Feb. 19, 2010. The entirecontents of each of the above-referenced patent applications are herebyexpressly incorporated herein by reference.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH OR DEVELOPMENT

This invention was made with government support under Contract NumberEY019309 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND

Abnormal or aberrant neovascularization is associated with a number ofdiseases and disorders, including but not limited to, cancer,inflammatory disease, macular degeneration and diabetic retinopathy(DR).

The Wnt signaling pathway plays a crucial role in neovascularization andmany other associated biological processes, including retinal vesseldevelopment and the inflammation process. Mutation of Wnt signalingpathway genes Frizzled-4 (Fz4) or LRP5 leads to inhibition of retinalangiogenesis in familial exudative vitreoretinopathy (FEVR) patients,while Fz4 knockout mice exhibit incomplete retinal vascularization.Moreover, VEGF is upregulated as a result of mutational activation ofWnt signaling in colon cancer and human endothelial cells. VEGF is apotent mediator of vascular permeability and angiogenesis, and is anestablished therapeutic target for a number of angiogenesis associateddiseases, including cancer and age related macular degeneration. Anumber of other angiogenic regulators are also Wnt target genesincluding, but not limited to, FGF18, endothelin-1, Cx43, uPAR, MMP7,and MMP3.

Among the aberrant neovascularization associated diseases, diabeticretinopathy (DR) is a very common complication of diabetes mellitus andone of the four common sight-threatening conditions in developedcountries. Almost 100% of patients with type I diabetes and 60% of typeII diabetic patients will develop some degrees of retinopathy in theirlifetime. Approximately 10% of diabetic patients develop a severe visualhandicap after 15 years of diabetes. DR is a chronic and progressivedisorder, primarily affecting retinal capillaries. Breakdown of theblood-retinal barrier is a common pathological change in patients withdiabetes and in streptozotocin (STZ)-induced diabetic animal models. Inthe early stages of DR, the retinal vascular permeability is increasedwithout the appearance of clinical retinopathy. Retinal vascular leakageand thickening of the retina lead to diabetic macular edema (DME). Inthe late stages of DR, over-proliferation of capillary endothelial cellsresults in retinal neovascularization (NV), the abnormal formation ofnew vessels from preexisting capillaries in the retina and vitreous.This, in turn, leads to proliferative diabetic retinopathy (PDR). Theabnormal angiogenesis can ultimately cause severe vitreous cavitybleeding and/or retinal detachment, resulting in severe vision loss.

It has been shown that multiple growth factors in the eye, such as butnot limited to, VEGF, bFGF, IGF-1, and PEDF, are implicated in thepathogenesis of DR. Alterations of these growth factors and theirreceptors in diabetes have been identified in both experimental andclinical studies. Increased VEGF levels are at least partly responsiblefor retinal vascular leakage, retinal vascular hyper-permeability andretinal NV in patients with DR. VEGF therefore plays an important rolein the development and pathogenesis of DR. The upregulated expression ofretinal VEGF and its receptors correlates with retinal NV in OIR.Inhibition of VEGF and VEGF receptors has been shown to prevent retinalNV in diabetic and OIR animal models.

Accumulating evidence indicates that the Wnt signaling pathway not onlymediates inflammation, i.e., TNF-alpha, NF-κB translocation and VEGF,but also regulates angiogenesis in the eye. Studies demonstrated thatboth Frizzled-4 (Fz4) and Lrp5/6 are expressed in adult murine retinalvasculature. Mutations in the Fz4 or LRP5 gene in the human lead toinhibition of normal retinal angiogenesis in familial exudativevitreoretinopathy (FEVR) patients, and Fz4 knockout (fz4^(−/−)) miceexhibited an incomplete retinal vascularization. Meanwhile, it has beenshown that seven β-catenin/TCF binding sites occur in the gene promoterfor VEGF-A. Under hypoxia conditions, HIF-1α competes with TCF-4 to forma new complex with β-catenin instead of β-catenin/TCF in the HIF-1α genepromoter region. Moreover, VEGF is upregulated as a result of mutationalactivation of the Wnt/β-catenin signaling in colon cancer cells and inhuman endothelial cells. A variety of other angiogenic regulators havepreviously been reported as Wnt target genes including but not limitedto, FGF18, endothelin-1, Cx43, uPAR, MMP7, and MMP3. Thus Wnts mayregulate angiogenesis through induction of multiple angiogenic genes.

The canonical pathway is initiated when a Wnt ligand binds to a memberof the Frizzled serpentine receptor family and its co-receptor LRP6 or aclose relative such as LRP5. When the Wnt-induced Fz-LRP6 complex forms,LRP6 will be phosphorylated at its PPPSP motif and is then capable ofbinding Axin in a phosphorylation-dependent manner to the plasmamembrane, thereby resulting in the inhibition of β-cateninphosphorylation and degradation. LRP6 is of critical importance in humandiseases. The LRP6 cytoplasmic domain is essential for Axin binding, andits deletion in LRP6 ΔC results in a dominant negative receptor thatbinds Wnt but is unable to bind Axin. The LRP6 extracellular domain hasauto-inhibitory activity, because its deletion in LRP6ΔN results in aconstitutively activated receptor that binds Axin in the absence of Wntligand.

As stated herein above, retinal NV is a major pathological featureleading to vision loss in DR. VEGF is a well-known key factor instimulating the retinal NV formation in the DR.

Therefore, there exists a great need for new and improved compositionsand methods for the inhibition of the Wnt signaling pathway. Suchcompositions and methods would be useful in the treatment and preventionof neovascularization-associated and/or Wnt signaling pathway associateddiseases, including but not limited to, inflammation, fibrosis,angiogenesis and/or tumorigenesis. The presently disclosed inventiveconcept(s) is directed to said compositions and methods, which overcomethe disadvantages and defects of the prior art.

BRIEF DESCRIPTION OF THE DRAWINGS

This patent or application file contains at least one drawing executedin color. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 illustrates activated Wnt signaling in the human retina with DR.Retinal sections from five non-DM donors and six diabetic donors withNPDR were immunostained with an antibody for β-catenin. The signal wasdeveloped with the diaminobenzidine method (brown color). Representativeretinal images from two non-DM (A and B) and two NPDR donors (C and D)showed more intensive signals of β-catenin in the inner retina andincreased β-catenin staining in the nuclei of the retinal cells from theDM-NPDR donors, compared with that from the non-DM subjects. Scalebar=20 μm. E: β-catenin signal was quantified by using morphometricanalysis software and expressed as arbitrary units (mean±SD). **P<0.01.

FIG. 2 depicts increased β-catenin levels in the retinas of Akita mice,STZ-induced diabetic rats, and OIR rats. The retinas from Akita mice at16 weeks of age, STZ-DM rats at 16 weeks following the STZ injection,OIR rats at the age of P16, and age-matched non-diabetic or normoxiccontrols were used for Western blot (A-C) and immunohistochemistry (D-Q)analyses of β-catenin. A-C: The same amount (50 μg) of retinal proteinsfrom each animal was blotted with an antibody specific for β-catenin.The membranes were stripped and reblotted with an antibody for β-actin.Each lane represents an individual animal. D-Q: Representative retinalsections from Akita mice (G-I) and their non-diabetic littermates (D-F),STZ-DM rats (M-O) and non-DM rats (J-L), OIR rats (Q), and age-matchednormal rats maintained under constant normoxia (P) were immunostainedwith an antibody for β-catenin. F, I, L, and O: The nucleus wascounterstained with 4′,6-diamidino-2-phenylindole (DAPI) (colored red)and merged with β-catenin signal. Red arrows (in I and O) indicate thenuclei showing green or orange color as a result of increased β-cateninsignal in the nuclei of diabetic retinas, while the white arrows (in Fand L) point to nuclei (red color) in non-diabetic retinas. Scale bar=20μm.

FIG. 3 illustrates up-regulated expression of LRP5/6 in the retinas ofSTZ-diabetic and OIR rats. A and B: The same amount of retinal proteins(100 μg) from three STZ-induced diabetic rats 16 weeks after the onsetof diabetes and age-matched non-diabetic rats (A), and four OIR rats andnormal rats at age of P16 (B) was used for Western blot analysis usingan antibody specific for LRP5/6 (Santa Cruz Biotechnology). The samemembranes were stripped and reblotted with an antibody for β-actin. C-F:Retinal sections from STZ-diabetic rats (D) and non-DM controls (C), andthose from OIR rats (F) and their normoxic controls (E) wereimmunostained with the antibody against LRP5/6 (green). The nuclei werecounterstained with DAPI (red). Original magnification, ×400.

FIG. 4 depicts induction of Wnt signaling by hypoxia and oxidativestress. A: RCEC were exposed to 2% oxygen and normoxia for 14 hours.Levels of total β-catenin were determined by Western blot analysis usingthe same amount (50 μg) of total proteins from each sample andnormalized to β-actin levels. Note that the blot represents twoindependent experiments. B-D: RCEC were treated with low glucose (LG; 5mmol/L glucose and 25 mmol/L mannitol, B), high glucose (HG, 30 mmol/Lglucose, C), and high glucose plus 10 μmol/L aminoguanidine (HG+AG, D)for 24 hours. The subcellular distribution of β-catenin was revealed byimmunocytochemistry by using the antibody for β-catenin. E: The sameamount of isolated nuclear proteins (50 μg) from each of the abovegroups was blotted with an antibody for β-catenin and normalized to TATAbox-binding protein (TBP) levels.

FIG. 5 illustrates that DKK1 ameliorates retinal inflammation, vascularleakage, and NV, and inhibits ROS generation. A: Various doses ofpurified DKK1 were injected into the vitreous of the right eye ofSTZ-diabetic rats at 16 weeks following the onset of diabetes, and thesame amounts of BSA were injected into the contralateral eyes forcontrols. Soluble ICAM-1 concentrations in the retina were measured byenzyme-linked immunosorbent assay, normalized by total proteinconcentrations, and expressed as ng per mg of proteins (means±SD, n=3).B: Purified DKK1 was injected into the vitreous of the right eye (1.2μg/eye) of STZ-diabetic rats at 16 weeks following the onset ofdiabetes, and the same amounts of BSA were injected into thecontralateral eyes for controls. Retinal vascular leakage was measured48 hours after the injection by using Evans blue as a tracer, normalizedby total protein concentrations, and expressed as μg of Evans blue permg of retinal proteins (means±SD, n=4). C and D: At the age of P14, theOIR rats received an intravitreal injection of DKK1 (1 μg/eye) into theright eye and the same amount of BSA into the contralateral eyes. Theretinas were harvested at P16, and the same amount of retinal proteins(20 μg) was loaded for Western blot analysis by using antibodiesspecific for COX2 (C) and VEGF (D), and normalized by β-actin levels. E:OIR rats at P14 received an intravitreal injection of DKK1 at doses asindicated. Retinal vascular leakage was measured at P16 by using Evansblue as a tracer, normalized by total protein concentrations, andexpressed as μg of Evans blue per mg of retinal proteins (means±SD,n=3). *P<0.05. F-J: OIR rats received an intravitreal injection of 2μg/eye DKK1, and BSA into the contralateral eyes at age of P14. At P18,retinal vasculature was visualized by fluorescein angiography on thewhole-mounted retina from the eyes injected with BSA (F and G) andinjected with DKK1 (H and I). Original magnification: ×12.5 (F and H);×100 (G and I). J: Preretinal vascular cells were counted on crossocular sections from the eyes injected with BSA and DKK1 (means±SD,n=5).

FIG. 6 illustrates that the Wnt pathway contributes to the oxidativestress and HIF-1 activation. A-D: DKK1 inhibits HIF-α activation:Primary RCEC were exposed to 5 mmol/L glucose and 25 mmol/L mannitol(A), 30 mmol/L glucose (B), TNF-α (C), and 30 mmol/L glucose with 1μg/ml DKK1 (D) for 4 hours. HIF-1α nuclear translocation was determinedby using immunocytochemistry with an anti-HIF-1α antibody. Scale bar=50μm. E: RCEC were exposed to low glucose (5 mmol/L glucose plus 25 mmol/Lmannitol) or high glucose (30 mmol/L glucose), or 1 μg/ml TNF-α in theabsence or presence of various concentrations of DKK1 (6.25 to 100nmol/L). Aminoguanidine (AG; 10 μmol/L) was used as a positive control.Intracellular ROS generation was measured and expressed as fluorescentunit per well (means±SD, n=3).

FIG. 7 depicts a Western blot analysis demonstrating that Anti-LRP6-1mAb inhibits LRP6 activity completely. RCECs were pretreated withAnti-LRP6-1 for 10 minutes, and then 20% Wnt3a-containing medium wasintroduced and cultured for another 16 hours. Western blotting analysisshowed that LRP6 phosphorylation was inhibited in a dose-dependentmanner, indicating LRP6 activity was blocked.

FIG. 8 illustrates that Anti-LRP6-1 mAb inhibits Wnt signaling pathwayactivation. RCEC cells were pretreated without and with Anti-LRP6-1 for10 minutes, and different concentration of Dkk1 were also used forpositive control. After pretreatment with Anti-LRP6-1 or Dkk1, RCECswere separately distributed to three different groups exposed toWnt3a-containing medium, hypoxia, or high glucose, respectively. Leftpanel: Western blotting analysis demonstrated that LRP6 phosphorylationwas inhibited. Right panel: TOPflash analysis of β-catenin showed thatAnti-LRP6-1 essentially completely inhibited β-catenin accumulation.

FIG. 9 graphically depicts that Anti-LRP6-1 inhibited proliferation ofendothelial cells. RCECs were seeded in 96-well tissue culture plates,and [¹⁴C]thymidine was added to the cells and mixed with differentconcentrations of Anti-LRP6-1 or DKK1. RCECs were then exposed to 1% O₂for 3 days, and the radioactivity incorporated into the cell monolayerwas determined and the percentage inhibition of the maximal signal wascalculated.

FIG. 10 graphically depicts that Anti-LRP6-1 decreased VEGFover-expression. RCECs were pretreated with Anti-LRP6-1 for 10 minutes,and exposed to hypoxia for 24 hours. The VEGF levels in the culturemedium were measured using ELISA. Anti-LRP6-1 significantly decreasedVEGF over-expressed in the endothelial cells that were exposed tohypoxic conditions.

FIG. 11 graphically depicts a comparison of the effects of Dkk1 andAnti-LRP6-1 on ROS generation induced by high glucose. RCEC were exposedto 30 mM glucose in the absence or presence of various concentrations ofDKK1 (6.25-100 nM) and Anti-LRP6-1 for 2 h. Intracellular ROS generationwas measured using CM-H₂DCFDA, and expressed as fluorescent unit perwell (means±SD, n=3).

FIG. 12 comprises photomicrographs that demonstrate that Anti-LRP6-1attenuates retinal vascular leukostasis in diabetic rats. STZ diabeticrats at 2 weeks after the onset of diabetes received an intravitrealinjection of 5 μM/eye of Anti-LRP6-1 in the treatment group and the samedose of Control mouse IgG and Dkk1 in the control group. 4 weeks afterthe injection, retinal vascular leukostasis was performed. The retinalvasculature and adherent leukocytes were stained by FITC-conjugatedconcanavalin A. A-D: Retinal vasculature and adherent leukocytes innon-diabetic rats (A), STZ-induced diabetic rats with IgG (B),STZ-induced diabetic rats injected with Dkk1 (C), and Anti-LRP6-1 (D).

FIG. 13 illustrates the effect of the Anti-LRP6-1 on ischemia-inducedretinal vascular leakage and VEGF over-expression. The Anti-LRP6-1 wasinjected intravitreally (10 μg/eye) into the OIR rats at P12. (A)Vascular permeability was measured at P16 using Evans blue method andnormalized by total retinal protein concentration (mean±SD, n=4). (B)The same amount of total retinal proteins (50 μg/rat) was immunoblottedwith an antibody for VEGF and normalized by β-actin levels.

FIG. 14 graphically depicts the effect of the Anti-LRP6-1 on AMD.Anti-LRP6-1 was injected intravitreally (10 μg/eye) into Vldlr^(−/−)mice. Retinal vascular permeability was measured 2 weeks after theinjection with Evans blue method and normalized by total retinal proteinconcentration (mean±SD, n=4).

FIG. 15 illustrates specificity of Anti-LRP6-1 on LRP6 ectodomain. A:HEK-293T cells were separately transfected with plasmids expressingLRP5-Flag and LRP6-Myc, with an empty vector as control. Forty-eighthours post-transfection, total cell lysates (50 μg) were applied forWestern blot analysis using Anti-LRP6-1 and an anti-Flag antibody (M2).B: The ability of Anti-LRP6-1 to recognize endogenous LRP6 in differentspecies. Total cell lysates (50 μg) from each cell line were applied forWestern blot analysis using Anti-LRP6-1. C: Conditioned media containingLDLRN-Myc, LRP5N-Myc and LRP6N-Myc, and purified recombinant peptides ofVLDLR-N-His, LRP6E1E2-His and LRP6E3E4-His were loaded for Western blotanalysis using Anti-LRP6-1, anti-His and anti-Myc antibodies. D:Immuno-localization of LRP6 in the retinal cryosections. FITC-labeledAnti-LRP6-1 was used to determine the cellular localization of LRP6 inthe retinas of rats (D1-D6) and mice (D7-D12). Non-specific mouse IgGwas labeled with FITC and used as a negative control (D1, D4, D7, D10).The sections were stained with FITC-Anti-LRP6-1 (D2, D5, D8, D11).FITC-Anti-LRP6-1 pre-absorbed with 10 μg/ml purified LRP6E1E2-Hisantigen was used for staining to confirm the specificity of theimmunosignals of LRP6 (D3, D6, D9, D12).

FIG. 16 depicts the inhibitory effect of the Anti-LRP6-1 on the Wntsignaling at the level of receptor-ligand interaction. A: hTERT-RPEcells at 70% confluence were serum-starved for 3 hours prior to Wntsignaling activation. Each well was pre-incubated for 30 minutes with 0,0.5, 5 and 50 μg/ml of Anti-LRP6-1; non-specific mouse IgG wassupplemented to reach total IgG concentrations to 50 μg/l in every well.Then, 25% Wnt3A conditioned media (Wnt3A) were added to the culturemedium with L cell CM (L) as a control. After 2 hours stimulation, equalamount of cell lysates (50 μg) were subjected to Western blot analysisusing antibodies for phosphorylated LRP6 (p-LRP6) (Ser1490) and fortotal LRP6. Cytosolic proteins (20 μg) were isolated and blotted with anantibody for cytosolic β-catenin (cyto-β-ctnn). B: hTERT-RPE cells weretransfected with TOPFLASH and control pRL-TK plasmids. The cells wereexposed to 25% WCM (Wnt3A) with various concentrations of Anti-LRP6-1for 16 hours, and then TCF/β-catenin activity was measured usingdual-luciferase assay and expressed as relative luciferase unit (RLU)(mean±SD, n=4, *P<0.05, **P<0.01). C: hTERT-RPE cells were transfectedwith TOPFLASH vectors and a Wnt1 expression plasmid. The vector withoutinsert was used as a control. At 4 hours post-transfection, cells weretreated with Anti-LRP6-1 at the indicated concentrations for 16 hours,and then luciferase activity was measured (mean±SD, n=4, †P<0.001). D:The cells were exposed to 25 mM LiCl to activate Wnt signaling, withNaCl as control. Equal amount of non-specific IgG or Anti-LRP6-1 (50μg/ml) were added to the cells and incubated for 16 hours, andLuciferase activity was measured (mean+SD, n=4, ‡P<0.0001, N.S: notstatistically significant).

FIG. 17 illustrates the inhibitory effect of the Anti-LRP6-1 on highglucose-induced canonical Wnt signaling. Retinal cells were exposed tohigh glucose media using 30 mmol/L D-glucose (HG) with 5 mmol/LD-glucose and 25 mmol/L mannitol (M) for osmotic control. A: hTERT-RPEcells were exposed to HG for different durations. Total cell lysateswere used to measure the levels of p-LRP6, total LRP6, and β-catenin. B:hTERT-RPE cells were exposed to high glucose for 6 hours with differentconcentrations of Anti-LRP6-1. Total cell lysates were used for Westernblot analysis to measure p-LRP6 and total LRP6; the cytosolic fractionwas used for measurement of cytosolic β-catenin (Cyto-β-ctnn). Levels ofcytosolic β-catenin from four independent Western blot analysis werequantified by densitometry and normalized by 0-actin levels (mean±SD,n=4, *P<0.05, †P<0.001). C: hTERT-RPE cells were exposed to high glucosefor 24 hours with 50 μg/ml Anti-LRP6-1 or IgG, followed by Western blotanalysis using antibodies specific for Cyclin D1 and c-myc. D & E: AMüller cell line (rMC-1) (D) and primary BRCEC (E) were exposed to HGfor 6 hours after 1 hour pre-incubation with Anti-LRP6-1. Cytosolicfraction (20 μg) was subjected to Western blot analysis for cytosolicβ-catenin and phospho-β-catenin (Ser33/37/Thr41).

FIG. 18 illustrates the inhibitory effect of Anti-LRP6-1 on highglucose-induced over-expression of angiogenic and inflammatory factors.hTERT-RPEs (A), rMC-1 (B), and BRCEC (C) were exposed to 30 mmol/LD-glucose (HG), with 5 mM glucose and 25 mM mannitole (M) as control,for 24 hours (A & C) or 48 hours (B) following 1 hour pre-incubation ofthe Anti-LRP6-1. Western blot analysis was performed using specificantibodies for ICAM-1, TNF-α and VEGF. Representative blots from atleast three independent experiments are shown. Levels were quantified bydensitometry and normalized by β-actin levels (mean±SD, n=3, *P<0.05,†P<0.001, ‡P<0.0001).

FIG. 19 illustrates the inhibitory effect of Anti-LRP6-1 on migration ofendothelial cells. A: Endothelial migration in the scratch wound model.BRCEC plated on the gelatin-coated plates were scratched and exposed tohigh glucose for 48 hours with 20 μg/ml Anti-LRP6-1 or non-specific IgG.Representative images of the scratch area are shown. B: Pictures ofthree different areas of each plate were taken and the surface areawithout migrated cells was measured using Image J (NIH) for thequantification. High glucose increased migration rate of BRCEC.Anti-LRP6-1 significantly suppressed migration of the BRCEC compared tonon-specific IgG (mean±SD, n=3, †P<0.001, *P<0.05). C: Tube formation:Matrigel was thawed on ice and spread evenly into a 24 well plate,followed by 30 minutes incubation at 37° C. BRCEC (2.5×10⁴) were seededinto each well and supplemented with PBS and 20 μg/ml non-specific IgGas negative controls and 20 μg/ml of Anti-LRP6-1. BRCEC were grown toform a tube for 12 hours and five fields from each well werephotographed. D: Quantification of the tube formation was performed bycounting the number of the branches in the fields. Anti-LRP6-1significantly suppressed tube formation of BRCEC compared tonon-specific IgG (mean±SD, n=3, †P<0.001).

FIG. 20 illustrates the inhibitory effect of Anti-LRP6-1 on the vascularleakage and inflammation in the OIR model. A: OIR rats received anintravitreal injection of 10 μg Anti-LRP6-1 per eye, and the same amountof non-specific IgG to the contralateral eyes at age of P12. Retinalvascular permeability was measured at P16 using Evans blue as a tracer,normalized by total retinal protein concentrations, and expressed as μgof Evans blue per mg of retinal proteins (means±SD, n=8, P<0.001).Dotted line indicates the basal level of vascular permeability inage-matched normal animals. B-D: The retinas were harvested at P16, andthe same amount of retinal proteins (50 μg) was loaded for Western blotanalysis using antibodies specific for LRP6, β-catenin, ICAM-1, VEGF andTNF-α (FIG. 20B), and normalized by β-actin levels (means±SD, n=3,*P<0.05, †P<0.001; FIG. 20C-D).

FIG. 21 illustrates the inhibitory effect of Anti-LRP6-1 on retinalvascular leakage and inflammation in the STZ-induced diabetic rats.STZ-induced diabetic rats at 2 weeks post-onset of diabetes received anintravitreal injection of Anti-LRP6-1 (20 μg) or the same amount ofnon-specific IgG as control. A: retinal vascular permeability wasmeasured at 1 week post-injection of the antibody using Evans blue as atracer, and normalized by total retinal protein concentrations(means±SD, n=6, P=0.00667). B: Adherent leukocytes were stained withFITC-concanavalin-A in non-diabetic rats, diabetic rats treated with IgGor Anti-LRP6-1 after the circulating leukocytes were removed by thoroughperfusion. The retinas were then flat-mounted, and the adherentleukocytes were visualized under fluorescence microscope. Representativeimages are shown in (B). Multiple leukocytes adherent to retinalvasculature were observed in the diabetic rat retinas and those with IgGinjection, but fewer in the Anti-LRP6-1 treated diabetic rats. Adherentleukocytes were counted in 4 random fields of each retina (mean±SD, n=5,‡P<0.0001). C: Two weeks after the intravitreal injection, the retinaswere dissected from non-diabetic (non-DM), diabetic (DM), and diabeticrats treated with IgG (DM-IgG) or Anti-LRP6-1 (DM-2F1) groups. The sameamount of retinal proteins was loaded for Western blot analysis tomeasure expression levels of LRP6, ICAM-1 and TNF-α (mean±SD, n=3,*P<0.05, **P<0.01, †P<0.001).

FIG. 22 illustrates inhibition of ICAM1 and CTGF expression byAnti-LRP6-1 under hypoxia. ARPE19 cells were exposed to 200 μM CoCl₂ inthe presence of various concentrations of Anti-LRP6-1 for 24 hours.Non-specific IgG (50 μg/ml) was used as control. Equal amount of thetotal cell lysates were loaded for Western blot analysis to measure theexpression level of ICAM-1 and CTGF and normalized by β-actin levels.

FIG. 23 illustrates that Anti-LRP6-1 decreases β-catenin levels in theeyecup with laser-induced CNV. Rats received laser photocoagulationfollowed by intravitreal injection of 20 μg/eye Anti-LRP6-1 ornon-specific rat IgG. At day 7 after the injection, the retina-choroidcomplex was dissected and β-catenin levels measured by Western blotanalysis. The same amount of proteins from normal eyes and that with CNVbut without the injection was blotted for comparison. The totalβ-catenin levels were quantified by densitometry and normalized toβ-actin levels (mean±SD, n=6). CNV showed up-regulated β-catenin levelswhich were decreased by Anti-LRP6-1 in the CNV model. Each lanerepresents an individual rat. *P<0.05.

FIG. 24 illustrates that Anti-LRP6-1 decreases numbers of Grade 4lesions in laser-induced CNV. Fluorescein angiography was performed 14days after the laser photocoagulation. Fundus images were captured. (A)The fundus image without fluorescein angiography; (B-D) representativefundus images with fluorescein angiography. (E) Grade 4 lesions werecounted and compared (mean±SD, n=10) *P<0.01.

FIG. 25 illustrates that Anti-LRP6-1 decreases CNV area. CNV was inducedby laser photocoagulation in rats. Anti-LRP6-1 (20 μg/eye) was injectedinto the vitreous with the same amount rat IgG as control at the sameday of the laser. Two weeks post-injection, CNV was visualized withfluorescein angiography. (A-C) Representative micrographs of CNV lesionsin an RPE-choroidal flat-mount in the CNV without treatment (A), withcontrol IgG (B) and with Anti-LRP6-1 (C). (D) The CNV areas weremeasured and compared (mean±SD, n=20). *P<0.05.

DETAILED DESCRIPTION OF THE INVENTIVE CONCEPT(S)

Before explaining at least one embodiment of the presently disclosedinventive concept(s) in detail by way of exemplary drawings,experimentation, results, and laboratory procedures, it is to beunderstood that the inventive concept(s) is not limited in itsapplication to the details of construction and the arrangement of thecomponents set forth in the following description or illustrated in thedrawings, experimentation and/or results. The inventive concept(s) iscapable of other embodiments or of being practiced or carried out invarious ways. As such, the language used herein is intended to be giventhe broadest possible scope and meaning; and the embodiments are meantto be exemplary—not exhaustive. Also, it is to be understood that thephraseology and terminology employed herein is for the purpose ofdescription and should not be regarded as limiting.

Unless otherwise defined herein, scientific and technical terms used inconnection with the presently disclosed inventive concept(s) shall havethe meanings that are commonly understood by those of ordinary skill inthe art. Further, unless otherwise required by context, singular termsshall include pluralities and plural terms shall include the singular.Generally, nomenclatures utilized in connection with, and techniques of,cell and tissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. See e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual (2^(nd) ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Coligan etal. Current Protocols in Immunology (Current Protocols, WileyInterscience (1994)), which are incorporated herein by reference. Thenomenclatures utilized in connection with, and the laboratory proceduresand techniques of, analytical chemistry, synthetic organic chemistry,and medicinal and pharmaceutical chemistry described herein are thosewell known and commonly used in the art. Standard techniques are usedfor chemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich the presently disclosed inventive concept(s) pertains. Allpublications and patent applications are herein incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of the presentlydisclosed inventive concept(s) have been described in terms ofparticular embodiments, it will be apparent to those of skill in the artthat variations may be applied to the compositions and/or methods and inthe steps or in the sequence of steps of the method described hereinwithout departing from the concept, spirit and scope of the inventiveconcept(s). All such similar substitutes and modifications apparent tothose skilled in the art are deemed to be within the spirit, scope andconcept of the inventive concept(s) as defined by the appended claims.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects. The use of the term “atleast one” will be understood to include one as well as any quantitymore than one, including but not limited to, 2, 3, 4, 5, 10, 15, 20, 30,40, 50, 100, etc. The term “at least one” may extend up to 100 or 1000or more, depending on the term to which it is attached; in addition, thequantities of 100/1000 are not to be considered limiting, as higherlimits may also produce satisfactory results.

The term “about” is used to indicate that a value includes the inherentvariation of error for the device, the method being employed todetermine the value and/or the variation that exists among studysubjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, MB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

The terms “peptide,” “polypeptide,” and “protein” are used herein torefer to a polymer of amino acid residues. The term “polypeptide” asused herein is a generic term to refer to native protein, proteinfragments, or analogs of a polypeptide sequence. Hence, native protein,protein fragments, and analogs are species of the polypeptide genus. Theterm “isolated peptide/polypeptide/protein” as used herein refers to apeptide/polypeptide/protein of cDNA, recombinant RNA, or syntheticorigin or some combination thereof, which by virtue of its origin, orsource of derivation, the “isolated peptide/polypeptide/protein”: (1) isnot associated with peptides/polypeptides/proteins found in nature, (2)is free of other peptides/polypeptides/proteins from the same source,e.g., free of murine proteins, (3) is expressed by a cell from adifferent species, and/or (4) does not occur in nature.

As used herein, the term “amino acid” embraces all molecules, whethernatural or synthetic, which include both an amino functionality and anacid functionality and capable of being included in a polymer ofnaturally-occurring amino acids. Exemplary amino acids includenaturally-occurring amino acids; analogs, derivatives, and congenersthereof; amino acid analogs having variant side chains; and allstereoisomers of any of any of the foregoing.

The terms “polynucleotide,” and “nucleic acid” are used interchangeably.They refer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof. Thefollowing are non-limiting examples of polynucleotides: coding ornon-coding regions of a gene or gene fragment, loci (locus) defined fromlinkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA,ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branchedpolynucleotides, plasmids, vectors, isolated DNA of any sequence,isolated RNA of any sequence, nucleic acid probes, and primers.

A polynucleotide may comprise modified nucleotides, such as methylatednucleotides and nucleotide analogs. If present, modifications to thenucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modified,such as by conjugation with a labeling component. The terms “isolatednucleic acid” and “isolated polynucleotide” are used interchangeably; anucleic acid or polynucleotide is considered “isolated” if it: (1) isnot associated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (2) is linked to apolynucleotide to which it is not linked in nature, or (3) does notoccur in nature as part of a larger sequence.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby bereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes. Such vectors are referredto herein as “recombinant expression vectors” (or simply, “expressionvectors”).

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolynucleotide or polypeptide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory orotherwise is naturally-occurring. The term “naturally-occurring” may beused interchangeably herein with the term “native.”

The term “selectively hybridize” referred to herein means to detectablyand specifically bind. Polynucleotides, oligonucleotides and fragmentsthereof encoding peptides/polypeptides/proteins in accordance with theinventive concept(s) selectively hybridize to nucleic acid strands underhybridization and wash conditions that minimize appreciable amounts ofdetectable binding to nonspecific nucleic acids. High stringencyconditions can be used to achieve selective hybridization conditions asknown in the art and discussed herein. Generally, the nucleic acidsequence homology between the polynucleotides, oligonucleotides, andfragments of the inventive concept(s) and a nucleic acid sequence ofinterest will be at least 80%, and more typically with increasinghomologies of at least 85%, 90%, 95%, 99%, and 100%. Two amino acidsequences are homologous if there is a partial or complete identitybetween their sequences. For example, 85% homology means that 85% of theamino acids are identical when the two sequences are aligned for maximummatching. Gaps (in either of the two sequences being matched) areallowed in maximizing matching; gap lengths of 5 or less are preferredwith 2 or less being more preferred. Alternatively and preferably, twoprotein sequences (or polypeptide sequences derived from them of atleast 30 amino acids in length) are homologous, as this term is usedherein, if they have an alignment score of at more than 5 (in standarddeviation units) using the program ALIGN with the mutation data matrixand a gap penalty of 6 or greater. See Dayhoff, M. O., in Atlas ofProtein Sequence and Structure, pp. 101-110 (Volume 5, NationalBiomedical Research Foundation (1972)) and Supplement 2 to this volume,pp. 1-10. The two sequences or parts thereof are more preferablyhomologous if their amino acids are greater than or equal to 50%identical when optimally aligned using the ALIGN program. The term“corresponds to” is used herein to mean that a polynucleotide sequenceis homologous (i.e., is identical, not strictly evolutionarily related)to all or a portion of a reference polynucleotide sequence, or that apolypeptide sequence is identical to a reference polypeptide sequence.In contradistinction, the term “complementary to” is used herein to meanthat the complementary sequence is homologous to all or a portion of areference polynucleotide sequence. For illustration, the nucleotidesequence “TATAC” corresponds to a reference sequence “TATAC” and iscomplementary to a reference sequence “GTATA.”

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotide or amino acid sequences: “referencesequence,” “comparison window,” “sequence identity,” “percentage ofsequence identity,” and “substantial identity.” A “reference sequence”is a defined sequence used as a basis for a sequence comparison; areference sequence may be a subset of a larger sequence, for example, asa segment of a full-length cDNA or gene sequence given in a sequencelisting or may comprise a complete cDNA or gene sequence. Generally, areference sequence is at least 18 nucleotides or 6 amino acids inlength, frequently at least 24 nucleotides or 8 amino acids in length,and often at least 48 nucleotides or 16 amino acids in length. Since twopolynucleotides or amino acid sequences may each (1) comprise a sequence(i.e., a portion of the complete polynucleotide or amino acid sequence)that is similar between the two molecules, and (2) may further comprisea sequence that is divergent between the two polynucleotides or aminoacid sequences, sequence comparisons between two (or more) molecules aretypically performed by comparing sequences of the two molecules over a“comparison window” to identify and compare local regions of sequencesimilarity. A “comparison window,” as used herein, refers to aconceptual segment of at least 18 contiguous nucleotide positions or 6amino acids wherein a polynucleotide sequence or amino acid sequence maybe compared to a reference sequence of at least 18 contiguousnucleotides or 6 amino acid sequences and wherein the portion of thepolynucleotide sequence in the comparison window may comprise additions,deletions, substitutions, and the like (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman (Adv.Appl. Math., 2:482 (1981)), by the homology alignment algorithm ofNeedleman and Wunsch (J. Mol. Biol., 48:443 (1970)), by the search forsimilarity method of Pearson and Lipman (Proc. Natl. Acad. Sci.(U.S.A.), 85:2444 (1988)), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package Release 7.0, (Genetics Computer Group, 575 Science Dr.,Madison, Wis.), Geneworks, or MacVector software packages, or byinspection, and the best alignment (i.e., resulting in the highestpercentage of homology over the comparison window) generated by thevarious methods is selected.

The term “sequence identity” means that two polynucleotide or amino acidsequences is identical (i.e., on a nucleotide-by-nucleotide orresidue-by-residue basis) over the comparison window. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I) or residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the comparison window (i.e., the windowsize), and multiplying the result by 100 to yield the percentage ofsequence identity. The terms “substantial identity” as used hereindenotes a characteristic of a polynucleotide or amino acid sequence,wherein the polynucleotide or amino acid comprises a sequence that hasat least 85 percent sequence identity, such as at least 90 to 95 percentsequence identity, or at least 99 percent sequence identity as comparedto a reference sequence over a comparison window of at least 18nucleotide (6 amino acid) positions, frequently over a window of atleast 24-48 nucleotide (8-16 amino acid) positions, wherein thepercentage of sequence identity is calculated by comparing the referencesequence to the sequence which may include deletions or additions whichtotal 20 percent or less of the reference sequence over the comparisonwindow. The reference sequence may be a subset of a larger sequence.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991)), which is incorporated herein by reference.Stereoisomers (e.g., D-amino acids) of the twenty conventional aminoacids, unnatural amino acids such as α-,α-disubstituted amino acids,N-alkyl amino acids, lactic acid, and other unconventional amino acidsmay also be suitable components for polypeptides of the presentlydisclosed inventive concept(s). Examples of unconventional amino acidsinclude: 4-hydroxyproline, α-carboxyglutamate, ε-N,N,N-trimethyllysine,ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and othersimilar amino acids and imino acids (e.g., 4-hydroxyproline). In thepolypeptide notation used herein, the lefthand direction is the aminoterminal direction and the righthand direction is the carboxy-terminaldirection, in accordance with standard usage and convention.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, such as at least 90 percent sequence identity, or atleast 95 percent sequence identity, or at least 99 percent sequenceidentity. Preferably, residue positions which are not identical differby conservative amino acid substitutions. Conservative amino acidsubstitutions refer to the interchangeability of residues having similarside chains. For example, a group of amino acids having aliphatic sidechains is glycine, alanine, valine, leucine, and isoleucine; a group ofamino acids having aliphatic-hydroxyl side chains is serine andthreonine; a group of amino acids having amide-containing side chains isasparagine and glutamine; a group of amino acids having aromatic sidechains is phenylalanine, tyrosine, and tryptophan; a group of aminoacids having basic side chains is lysine, arginine, and histidine; and agroup of amino acids having sulfur-containing side chains is cysteineand methionine. Preferred conservative amino acids substitution groupsare: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamic-aspartic, and asparagine-glutamine.

The term “variant” of a reference polypeptide refers to a polypeptidehaving one or more amino acid substitutions, deletions or insertionsrelative to the reference polypeptide. An amino acid substitution may be“conservative” or “non-conservative.” A “conservative” amino acidsubstitution refers to the substitution of an amino acid in apolypeptide with another amino acid having similar properties, such asbut not limited to, size and charge. Conservative replacements are thosethat take place within a family of amino acids that are related in theirside chains. Genetically encoded amino acids are generally divided intofamilies: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine,histidine; (3) nonpolar=alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine. Moreparticular families are: serine and threonine are aliphatic-hydroxyfamily; asparagine and glutamine are an amide-containing family;alanine, valine, leucine and isoleucine are an aliphatic family; andphenylalanine, tryptophan, and tyrosine are an aromatic family. Forexample, it is reasonable to expect that an isolated replacement of aleucine with an isoleucine or valine, an aspartate with a glutamate, athreonine with a serine, or a similar replacement of an amino acid witha structurally related amino acid will not have a major effect on thebinding or properties of the resulting molecule, especially if thereplacement does not involve an amino acid within a framework site.Whether an amino acid change results in a functional peptide can readilybe determined by assaying the specific activity of the polypeptidederivative. Fragments or analogs of antibodies or immunoglobulinmolecules can be readily prepared by those of ordinary skill in the art.Preferred amino- and carboxy-termini of fragments or analogs occur nearboundaries of functional domains. Structural and functional domains canbe identified by comparison of the nucleotide and/or amino acid sequencedata to public or proprietary sequence databases. Preferably,computerized comparison methods are used to identify sequence motifs orpredicted protein conformation domains that occur in other proteins ofknown structure and/or function. Methods to identify protein sequencesthat fold into a known three-dimensional structure are known (Bowie etal., Science, 253:164 (1991)). Thus, the foregoing examples demonstratethat those of skill in the art can recognize sequence motifs andstructural conformations that may be used to define structural andfunctional domains in accordance with the presently disclosed inventiveconcept(s).

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (5) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmutations of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally-occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure© (Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et al. (Nature 354:105 (1991)), which are each incorporatedherein by reference.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino-terminal and/or carboxy-terminal deletion, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the naturally-occurring sequence. A polypeptide fragmentmay be any length that is less than the length of the referencepolypeptide.

The term “antibody” is used in the broadest sense, and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired biological activity. Thus, the terms “Antibody” or “antibodypeptide(s)” refer to a full length immunoglobulin molecule (i.e., anintact antibody), or a binding fragment thereof that competes with theintact antibody for specific antigen binding. Binding fragments may beproduced by recombinant DNA techniques, or by enzymatic or chemicalcleavage of intact antibodies. Binding fragments include Fab, Fab′,F(ab′)₂, Fv, scFv, disulfide linked Fv, Fd, diabodies, single-chainantibodies, single domain antibodies (such as but not limited to,NANOBODIES®) and other antibody fragments that retain at least a portionof the variable region of an intact antibody. See, e.g., Hudson et al.(Nature Med., 9:129-134 (2003)).

The term “antigen binding fragment” or “antigen-binding portion” of anantibody, as used herein, refers to one or more fragments of an antibodythat retain the ability to bind to an antigen. The antigen-bindingfunction of an antibody can be performed by fragments of an intactantibody. Examples of binding fragments encompassed within the term“antigen-binding fragment” of an antibody include but are not limitedto, Fab, Fab′, F(ab′)2, Fv, scFv, disulfide linked Fv, Fd, diabodies,single-chain antibodies, single domain antibodies (such as but notlimited to, NANOBODIES®), isolated CDRH3, and other antibody fragmentsthat retain at least a portion of the variable region of an intactantibody. These antibody fragments are obtained using conventionalrecombinant and/or enzymatic techniques and are screened for antigenbinding in the same manner as intact antibodies.

The terms “CDR,” and its plural “CDRs,” refer to a complementaritydetermining region (CDR) of an antibody or antibody fragment, whichdetermine the binding character of an antibody or antibody fragment. Inmost instances, three CDRs are present in a light chain variable region(CDRL1, CDRL2 and CDRL3) and three CDRs are present in a heavy chainvariable region (CDRH1, CDRH2 and CDRH3). CDRs contribute to thefunctional activity of an antibody molecule and are separated by aminoacid sequences that comprise scaffolding or framework regions. Among thevarious CDRs, the CDR3 sequences, and particularly CDRH3, are the mostdiverse and therefore have the strongest contribution to antibodyspecificity. There are at least two techniques for determining CDRs: (1)an approach based on cross-species sequence variability (i.e., Kabat etal., Sequences of Proteins of Immunological Interest (National Instituteof Health, Bethesda, Md. (1987), incorporated by reference in itsentirety); and (2) an approach based on crystallographic studies ofantigen-antibody complexes (Chothia et al., Nature, 342:877 (1989),incorporated by reference in its entirety).

The term “epitope” includes any protein determinant capable of specificbinding to an immunoglobulin or T-cell receptor. In certain embodiments,an epitope is a region of an antigen that is specifically bound by anantibody. Epitopic determinants usually include chemically activesurface groupings of molecules such as amino acids, sugar side chains,phosphoryl, or sulfonyl groups. In certain embodiments, an epitope mayhave specific three dimensional structural characteristics (e.g., a“conformational epitope”), as well as specific charge characteristics.

An epitope is defined as “the same” as another epitope if a particularantibody specifically binds to both epitopes. In certain embodiments,polypeptides having different primary amino acid sequences may compriseepitopes that are the same. In certain embodiments, epitopes that arethe same may have different primary amino acid sequences. Differentantibodies are said to bind to the same epitope if they compete forspecific binding to that epitope.

An antibody “specifically binds” an antigen when it preferentiallyrecognizes the antigen in a complex mixture of proteins and/ormacromolecules. In certain embodiments, an antibody comprises anantigen-binding site that specifically binds to a particular epitope. Incertain such embodiments, the antibody is capable of binding differentantigens so long as the different antigens comprise that particularepitope or closely related epitopes. In certain instances, for example,homologous proteins from different species may comprise the sameepitope. In certain embodiments, an antibody specifically binds to anantigen with a dissociation constant of no greater than 10⁻⁶ M, 10⁻⁷ M,10⁻⁸ M or 10⁻⁹ M. When an antibody specifically binds to a receptor orligand (i.e., counterreceptor), it may substantially inhibit adhesion ofthe receptor to the ligand. As used herein, an antibody substantiallyinhibits adhesion of a receptor to a ligand when an excess of antibodyreduces the quantity of receptor bound to ligand by at least about 20%,40%, 60% or 80%, 85%, or 90% (as measured in an in vitro competitivebinding assay).

An “isolated” antibody is one which has been separated and/or recoveredfrom a component of the environment in which it was produced.Contaminant components of its production environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In certain embodiments, the antibody will bepurified as measurable by at least three different methods: 1) togreater than 50% by weight of antibody as determined by the Lowrymethod, such as more than 75% by weight, or more than 85% by weight, ormore than 95% by weight, or more than 99% by weight; 2) to a degreesufficient to obtain at least 10 residues of N-terminal or internalamino acid sequence by use of a spinning cup sequentator, such as atleast 15 residues of sequence; or 3) to homogeneity by SDS-PAGE underreducing or non-reducing conditions using Coomasie blue or, preferably,silver stain. Isolated antibody includes the antibody in situ withinrecombinant cells since at least one component of the environment inwhich the antibody is produced will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step. Inaddition, the “isolated antibody” is substantially free of otherantibodies having different antigenic specificities. An isolatedantibody may, however, have some cross-reactivity to other, relatedantigens.

The term “antibody mutant” refers to an amino acid sequence variant ofan antibody wherein one or more of the amino acid residues have beenmodified. Such mutants necessarily have less than 100% sequence identityor similarity with the amino acid sequence having at least 75% aminoacid sequence identity or similarity with the amino acid sequence ofeither the heavy or light chain variable domain of the antibody, such asat least 80%, or at least 85%, or at least 90%, or at least 95%.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies thatspecifically bind to the same epitope, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts. In contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that in one method of production they may be synthesizedby a hybridoma culture, and thus are uncontaminated by otherimmunoglobulins. The modifier “monoclonal” indicates the character ofthe antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, in oneembodiment, the monoclonal antibodies produced in accordance with thepresently disclosed inventive concept(s) may be made by the hybridomamethod first described by Kohler and Milstein (Nature, 256:495 (1975)).

The monoclonal antibodies utilized in accordance with the presentlydisclosed inventive concept(s) may be produced by any methodology knownin the art including, but not limited to, a result of a deliberateimmunization protocol; a result of an immune response that results inthe production of antibodies naturally in the course of a disease orcancer; phage-derived antibodies; and the like. In addition to thehybridoma production method listed above, the monoclonal antibodies ofthe presently disclosed inventive concept(s) may be produced by othervarious methods such as, but not limited to, recombinant DNA methods(see, e.g., U.S. Pat. No. 4,816,567); isolation of antibody fragmentsfrom a phage display library (see, e.g., Clackson et al., Nature, 352:624-628 (1991); and Marks et al., J. Mol. Biol., 222:581-597 (1991)); aswell as various other monoclonal antibody production techniques (see,e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual (ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.)).

Once the antibodies have been obtained, for example, once individual Bcells have been identified and/or monoclonal antibodies have beenproduced, the sequences encoding the variable regions of theseantibodies can be obtained. The variable region sequences can, forexample, be obtained by first sequencing the antibody protein producedby the hybridoma, B-cell or phage and determining the encoding nucleicacid sequence. In one embodiment, the immunoglobulin variable region (VHand VL) DNA or cDNA may be sequenced instead. Where the antibody isderived from a hybridoma cell line or isolated B-cell, the cDNAsencoding the variable regions may be amplified using PCR by, forexample, the methods described in Babcook et al. (Proc. Natl. Acad. Sci.USA, 93:7843-7848 (1996)), and in PCT Publication No. WO 92/02551. Thecontents of both references are expressly incorporated herein byreference in their entirety.

A “chimeric” antibody refers to an antibody made up of components fromat least two different sources. In certain embodiments, a chimericantibody comprises a portion of an antibody derived from a first speciesfused to another molecule, e.g., a portion of an antibody derived from asecond species. In certain such embodiments, a chimeric antibodycomprises a portion of an antibody derived from a non-human animal fusedto a portion of an antibody derived from a human. In certain suchembodiments, a chimeric antibody comprises all or a portion of avariable region of an antibody derived from one animal fused to aportion of an antibody from a second animal. For example but not by wayof limitation, a chimeric antibody may comprise all or portion of avariable region of an antibody derived from a non-human animal fused toa constant region of an antibody derived from a human.

Utilization of the monoclonal antibodies of the presently disclosedinventive concept(s) may require administration thereof to a subject,such as but not limited to, a human. However, when the monoclonalantibodies are produced in a non-human animal, such as a rodent,administration of such antibodies to a human patient will normallyelicit an immune response, wherein the immune response is directedtowards the sequence of the antibodies. Such reactions limit theduration and effectiveness of such a therapy. In order to overcome suchproblem, the monoclonal antibodies of the presently disclosed inventiveconcept(s) are “humanized,” that is, the antibodies are engineered suchthat one or more antigenic portions thereof are removed and likeportions of a human antibody are substituted therefore, while theantibodies' affinity for the desired epitope is retained. Thisengineering may only involve a few amino acids, or may include entireframework regions of the antibody, leaving only the complementaritydetermining regions of the antibody intact. Several methods ofhumanizing antibodies are known in the art and are disclosed in U.S.Pat. No. 6,180,370, issued to Queen et al. on Jan. 30, 2001; U.S. Pat.No. 6,054,927, issued to Brickell on Apr. 25, 2000; U.S. Pat. No.5,869,619, issued to Studnicka on Feb. 9, 1999; U.S. Pat. No. 5,861,155,issued to Lin on Jan. 19, 1999; U.S. Pat. No. 5,712,120, issued toRodriquez et al. on Jan. 27, 1998; and U.S. Pat. No. 4,816,567, issuedto Cabilly et al. on Mar. 28, 1989, the Specifications of which are allhereby expressly incorporated herein by reference in their entirety.

As mentioned above, a “humanized” antibody refers to a non-humanantibody that has been modified so that it more closely matches (inamino acid sequence) a human antibody. A humanized antibody is thus atype of chimeric antibody. As described above, antibodies interact withtarget antigens predominantly through amino acid residues that arelocated in the heavy and light chain complementarity determining regions(CDRs). For this reason, the amino acid sequences within CDRs are morediverse between individual antibodies than sequences outside of CDRs.Because CDR sequences are responsible for most antibody-antigeninteractions, it is possible to express recombinant antibodies thatmimic the properties of specific, naturally occurring antibodies byconstructing expression vectors in which the CDR sequences from thenaturally occurring antibody are grafted into framework sequences from adifferent antibody with different properties, such as human antibodyframework regions. Such framework sequences can be obtained from publicDNA databases or published references that include germline antibodygene sequences. For example, germline DNA sequences for human heavy andlight chain variable region genes can be found in the “VBase” humangermline sequence database (available on the Internet atwww.mrc-cpe.cam.ac.uk/vbase), as well as in Kabat, E. A., et al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242;Tomlinson, I. M., et al. (1992) “The Repertoire of Human Germline VHSequences Reveals about Fifty Groups of VH Segments with DifferentHypervariable Loops” J. Mol. Biol. 227:776-798; and Cox, J. P. L. et al.(1994) “A Directory of Human Germ-line VH Segments Reveals a Strong Biasin their Usage” Eur. J. Immunol. 24:827-836; the contents of each ofwhich are expressly incorporated herein by reference.

Humanized forms of antibodies are chimeric immunoglobulins,immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′,F(ab′)₂, or other antigen-binding subsequences of antibodies) that areprincipally comprised of the sequence of a human immunoglobulin, andcontain minimal sequence derived from a non-human immunoglobulin.Humanization can be performed following the method of Winter andco-workers (Jones et al., 1986; Riechmann et al., 1988; Verhoeyen etal., 1988), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody. (See also U.S. Pat. No.5,225,539.) In some instances, F_(v) framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies can also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of theframework regions are those of a human immunoglobulin consensussequence. The humanized antibody optimally also will comprise at least aportion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; andPresta, 1992).

The prior art is filled with published articles relating to thegeneration or use of humanized antibodies. Many of these studies teachuseful examples of protocols that can be utilized with the presentlydisclosed inventive concept(s), such as but not limited to, Sandborn etal., Gatroenterology, 120:1330 (2001); Mihara et al., Clin. Immunol.,98:319 (2001); Yenari et al., Neurol. Res., 23:72 (2001); Morales etal., Nucl. Med. Biol., 27:199 (2000); Richards et al., Cancer Res.,59:2096 (1999); Yenari et al., Exp. Neurol., 153:223 (1998); andShinkura et al., Anticancer Res., 18:1217 (1998), all of which areexpressly incorporated in their entirety by reference. However, it is tobe understood that the presently disclosed inventive concept(s) is notlimited to the treatment protocols described above, and other treatmentprotocols which are known to a person of ordinary skill in the art maybe utilized in the methods of the presently disclosed inventiveconcept(s).

The presently disclosed inventive concept(s) further includes the use offully human monoclonal antibodies. Fully human antibodies essentiallyrelate to antibody molecules in which the entire sequence of both thelight chain and the heavy chain, including the CDRs, arise from humangenes. Such antibodies are termed “human antibodies” or “fully humanantibodies” herein. “Human antibodies” contain human antibody sequencesand do not contain antibody sequences from a non-human animal. Incertain embodiments, a human antibody may further contain syntheticsequences not found in native antibodies. The term is not limited by themanner in which the antibodies are made.

Human monoclonal antibodies may be prepared by the trioma technique; thehuman B-cell hybridoma technique (see Kozbor et al., Hybridoma, 2:7(1983)) and the EBV hybridoma technique to produce human monoclonalantibodies (see Cole et al., PNAS, 82:859 (1985)). Human monoclonalantibodies may be utilized in the practice of the presently disclosedinventive concept(s) and may be produced by using human hybridomas (seeCote et al. PNAS, 80:2026 (1983)) or by transforming human B-cells withEpstein Barr Virus in vitro (see Cole et al., 1985).

In addition, human antibodies can be made by introducing humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and antibody repertoire. This approach isdescribed, for example but not by way of limitation, in U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and inMarks et al., J Biol. Chem., 267:16007 (1992); Lonberg et al., Nature,368:856 (1994); Morrison, 1994; Fishwild et al., Nature Biotechnol.,14:845 (1996); Neuberger, Nat. Biotechnol., 14:826 (1996); and Lonbergand Huszar, Int Rev Immunol., 13:65 (1995).

Human antibodies may additionally be produced using transgenic nonhumananimals which are modified so as to produce fully human antibodiesrather than the animal's endogenous antibodies in response to challengeby an antigen. (See PCT Publication No. WO 94/02602). The endogenousgenes encoding the heavy and light immunoglobulin chains in the nonhumanhost have been incapacitated, and active loci encoding human heavy andlight chain immunoglobulins are inserted into the host's genome. Thehuman genes are incorporated, for example, using yeast artificialchromosomes containing the requisite human DNA segments. An animal whichprovides all the desired modifications is then obtained as progeny bycrossbreeding intermediate transgenic animals containing fewer than thefull complement of the modifications. One embodiment of such a nonhumananimal is a mouse, and is termed the XENOMOUSE™ as disclosed in PCTPublication Nos. WO 96/33735 and WO 96/34096. This animal produces Bcells which secrete fully human immunoglobulins. The antibodies can beobtained directly from the animal after immunization with an immunogenof interest, as, for example, a preparation of a polyclonal antibody, oralternatively from immortalized B cells derived from the animal, such ashybridomas producing monoclonal antibodies. Additionally, the genesencoding the immunoglobulins with human variable regions can berecovered and expressed to obtain the antibodies directly, or can befurther modified to obtain analogs of antibodies such as, for example,single chain Fv molecules.

An example of a method of producing a nonhuman host, exemplified as amouse, lacking expression of an endogenous immunoglobulin heavy chain isdisclosed in U.S. Pat. No. 5,939,598, issued to Kucherlapati et al. onAug. 17, 1999, and incorporated herein by reference. It can be obtainedby a method including deleting the J segment genes from at least oneendogenous heavy chain locus in an embryonic stem cell to preventrearrangement of the locus and to prevent formation of a transcript of arearranged immunoglobulin heavy chain locus, the deletion being effectedby a targeting vector containing a gene encoding a selectable marker;and producing from the embryonic stem cell a transgenic mouse whosesomatic and germ cells contain the gene encoding the selectable marker.

A method for producing an antibody of interest, such as a humanantibody, is disclosed in U.S. Pat. No. 5,916,771, issued to Hori et al.on Jun. 29, 1999, and incorporated herein by reference. It includesintroducing an expression vector that contains a nucleotide sequenceencoding a heavy chain into one mammalian host cell in culture,introducing an expression vector containing a nucleotide sequenceencoding a light chain into another mammalian host cell, and fusing thetwo cells to form a hybrid cell. The hybrid cell expresses an antibodycontaining the heavy chain and the light chain.

The term “neutralizing antibody” or “antibody that neutralizes” refersto an antibody that reduces at least one activity of a polypeptidecomprising the epitope to which the antibody specifically binds. Incertain embodiments, a neutralizing antibody reduces an activity invitro and/or in vivo.

The term “antigen-binding site” refers to a portion of an antibodycapable of specifically binding an antigen. In certain embodiments, anantigen-binding site is provided by one or more antibody variableregions.

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition). Generally, asubstantially pure composition will comprise more than about 50% percentof all macromolecular species present in the composition, such as morethan about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 99%. In oneembodiment, the object species is purified to essential homogeneity(contaminant species cannot be detected in the composition byconventional detection methods) wherein the composition consistsessentially of a single macromolecular species.

The term “agent” refers to a chemical compound, a mixture of chemicalcompounds, a biological macromolecule, or an extract made frombiological materials. In certain embodiments, the “agent” may be amonoclonal antibody in accordance with the presently disclosed inventiveconcept(s).

The term “antagonist” refers to an agent that reduces an activity of aprotein/enzyme.

The term “agonist” refers to an agent that increases an activity of aprotein/enzyme.

The term “patient” includes human and veterinary subjects. In certainembodiments, a patient is a mammal. In certain other embodiments, thepatient is a human.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include, but are notlimited to, individuals already having a particular condition ordisorder as well as individuals who are at risk of acquiring aparticular condition or disorder (e.g., those needingprophylactic/preventative measures). The term “treating” refers toadministering an agent to a patient for therapeutic and/orprophylactic/preventative purposes.

A “therapeutic agent” refers to an agent that may be administered invivo to bring about a therapeutic and/or prophylactic/preventativeeffect.

A “therapeutic antibody” refers to an antibody that may be administeredin vivo to bring about a therapeutic and/or prophylactic/preventativeeffect.

A “disorder” is any condition that would benefit from treatment with thepolypeptide. This includes chronic and acute disorders or diseasesincluding those pathological conditions which predispose the mammal tothe disorder in question.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of include but are not limited to,carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particularexamples of such cancers include squamous cell cancer, small-cell lungcancer, non-small cell lung cancer, gastrointestinal cancer, pancreaticcancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer,bladder cancer, hepatoma, breast cancer, colon cancer, colorectalcancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer,renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma, and various types of head and neck cancer.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including human, domestic and farm animals, nonhuman primates,and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.

The term “effective amount” refers to an amount of a biologically activemolecule or conjugate or derivative thereof sufficient to exhibit adetectable therapeutic effect without undue adverse side effects (suchas toxicity, irritation and allergic response) commensurate with areasonable benefit/risk ratio when used in the manner of the presentlydisclosed inventive concept(s). The therapeutic effect may include, forexample but not by way of limitation, inhibiting and/or neutralizing atleast one activity of LRP6. The effective amount for a patient willdepend upon the type of patient, the patient's size and health, thenature and severity of the condition to be treated, the method ofadministration, the duration of treatment, the nature of concurrenttherapy (if any), the specific formulations employed, and the like.Thus, it is not possible to specify an exact effective amount inadvance. However, the effective amount for a given situation can bedetermined by one of ordinary skill in the art using routineexperimentation based on the information provided herein.

As used herein, the term “concurrent therapy” is used interchangeablywith the terms “combination therapy” and “adjunct therapy,” and will beunderstood to mean that the patient in need of treatment is treated orgiven another drug for the disease/disorder in conjunction with thecompositions of the presently disclosed inventive concept(s). Thisconcurrent therapy can be sequential therapy where the patient istreated first with one drug and then the other, or the two drugs aregiven simultaneously.

The term “pharmaceutically acceptable” refers to compounds andcompositions which are suitable for administration to humans and/oranimals without undue adverse side effects such as toxicity, irritationand/or allergic response commensurate with a reasonable benefit/riskratio.

By “biologically active” is meant the ability to modify thephysiological system of an organism. A molecule can be biologicallyactive through its own functionalities, or may be biologically activebased on its ability to activate or inhibit molecules having their ownbiological activity.

The compositions of the presently disclosed inventive concept(s) may beadministered to a patient by any method known in the art, including butnot limited to, oral, topical, transdermal, parenteral, subcutaneous,intranasal, intramuscular, intraperitoneal, intravitreal, andintravenous routes, including both local and systemic applications. Inaddition, the compounds of the presently disclosed inventive concept(s)may be designed to provide delayed, controlled or sustained releaseusing formulation techniques which are well known in the art.

The term “Wnt” or the plural “Wnts” as used herein will be understood torefer to a group of secreted, cysteine-rich glycoproteins which bind toa co-receptor complex of frizzled (Fz) receptors and low-densitylipoprotein receptor-related proteins 5 or 6 (LRP5/6) and regulateexpression of a number of target genes through an intracellularsignaling pathway, namely the Wnt pathway. In humans, the Wnts includeWNT1, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B,WNT8A, WNT8B, WNT9A, and WNT9B. In the absence of Wnt ligands,β-catenin, a down-stream effector of the canonical Wnt pathway, isphosphorylated by a protein complex containing glycogen synthasekinase-3β (GSK-3β). The phosphorylated β-catenin is constantly degraded,to prevent its accumulation. Upon binding of certain Wnts to theFz-LRP5/6 co-receptors, phosphorylation of β-catenin is inhibited, whichprevents the degradation of β-catenin and results in its accumulation.β-catenin is then translocated into the nucleus, where it associateswith T cell factor for DNA binding and thus regulates expression oftarget genes including but not limited to VEGF.

The term “LRP” will be understood to refer to “low-density lipoproteinreceptor-related proteins.” Human LRP6 is represented by SEQ ID NO: 1.LRP5/6 is known to play a critical role in Wnt/β-catenin signaling. Uponbinding with Wnt ligands, LRP6 dimerizes with Fz receptor, which is thefirst and essential step in activation of the Wnt pathway. Thecytoplasmic domain of LRP6 has multiple modular phosphorylation sites,and phosphorylation of LRP6 is an essential event for activation of thecanonical Wnt pathway, as the phosphorylation of LRP6 promotes therecruitment of the scaffold protein Axin, and thus activates thecanonical Wnt pathway.

The monoclonal antibodies described herein are characterized, in part,by functional and/or structural features of the antibodies.

The presently disclosed inventive concept(s) is related to an isolatedmonoclonal antibody (or antigen binding fragment thereof) thatspecifically binds LRP6 protein, as well as compositions comprisingsame. In one embodiment, the monoclonal antibody specifically binds thehuman LRP6 protein; in a further embodiment, the monoclonal antibodyspecifically binds an epitope in the amino acid sequence of SEQ ID NO:1.In yet another embodiment, the monoclonal antibody specifically binds anextracellular domain of LRP6; in a yet further embodiment, themonoclonal antibody specifically binds an epitope in the amino acidsequence of SEQ ID NO:2. In another embodiment, the monoclonal antibodyspecifically binds a ligand-binding domain of LRP6. In yet anotherembodiment, the monoclonal antibody specifically binds an epitope in atleast one of the first and second beta-propeller regions (E1E2 domains)of LRP6. In yet a further embodiment, the monoclonal antibodyspecifically binds an epitope in at least a portion of the E2 domain ofLRP6; in yet another further embodiment, the monoclonal antibodyspecifically binds an epitope in the amino acid sequence of SEQ ID NO:3.

Standard assays to evaluate the binding ability of the antibodies areknown in the art, including, for example, ELISAs, Western blots and RIAsand suitable assays are described in the Examples. The binding kinetics(e.g., binding affinity) of the antibodies also can be assessed bystandard assays known in the art, such as by Biacore analysis. In someembodiments, the antibodies described herein bind to SEQ ID NO: 1, SEQID NO: 2 and/or SEQ ID NO: 3 with a dissociation constant of less thanor equal to 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, or 10⁻⁹ M. In one embodiment, theantibody binds to the LRP6 extracellular domain with a dissociationconstant of less than or equal to about 10⁻⁷ M.

The presently disclosed inventive concept(s) is also directed to thehybridoma HLS2F1, ATCC accession number PTA-10663, as well ascompositions comprising same. Said hybridoma was deposited with theAmerican Type Culture Collection Patent Depository (10801 UniversityBoulevard, Manassas, Va. 20110-2209) on Feb. 18, 2010, under the termsof the Budapest Treaty. All restrictions on the availability to thepublic of the deposited material will be irrevocably removed upon thegranting of a patent directed to said mAb, and the deposit will bemaintained for 30 years or 5 years after the most recent request(whichever is later). The presently disclosed inventive concept(s) isalso directed to an isolated monoclonal antibody produced by saidhybridoma. The mAb produced by said deposited hybridoma is a murine IgG2antibody and will herein after be referred to as Anti-LRP6-1. Inaddition, the presently disclosed inventive concept(s) is also directedto a cell of hybridoma HLS2F1, ATCC accession number PTA-10663, as wellas compositions comprising same.

The presently disclosed inventive concept(s) is also directed to anisolated monoclonal antibody (or antigen binding fragment thereof) thatbinds to the same epitope as any of the monoclonal antibodies describedherein above, as well as compositions comprising same. In oneembodiment, the mAb binds to the same epitope as Anti-LRP6-1. In anotherembodiment, the mAb binds to the same epitope as the antibody producedby hybridoma HLS2F1, ATCC accession number PTA-10663. Such antibodiescan be identified based on their ability to cross-compete withanti-LRP6-1 in standard LRP6 extracellular domain binding assays. Theability of a test antibody to inhibit the binding of anti-LRP6-1 to theLRP6 extracellular domain demonstrates that the test antibody cancompete with anti-LRP6-1 for binding to the LRP6 extracellular domainand thus binds to the same epitope on the LRP6 extracellular domain asanti-LRP6-1.

The presently disclosed inventive concept(s) is also directed to anisolated monoclonal antibody that specifically binds to a sequence thatis at least 80% identical to any of SEQ ID NOS:1-3, including a sequencethat is at least 85% identical to any of SEQ ID NOS:1-3, a sequence thatis at least 90% identical to any of SEQ ID NOS:1-3, and a sequence thatis at least 95% identical to any of SEQ ID NOS:1-3, and compositionscomprising said isolated monoclonal antibody. In addition, the presentlydisclosed inventive concept(s) is also directed to an isolatedmonoclonal antibody having an amino acid sequence that is at least 80%identical (such as at least 85% identical, 90% identical, or 95%identical) to the amino acid sequence of the monoclonal antibodyproduced by the hybridoma deposited with the ATCC, as described indetail herein above.

Anti-LRP6-1 (the mAb produced by the deposited hybridoma as described indetail herein above, also referred to interchangeably as “mAb2F1”)comprises a heavy chain variable domain encoded by the nucleotidesequence of SEQ ID NO:4 and having an amino acid sequence as set forthin SEQ ID NO:5. Anti-LRP6-1 also comprises a light chain variable domainencoded by the nucleotide sequence of SEQ ID NO:6 and having an aminoacid sequence as set forth in SEQ ID NO:7. The heavy chain comprisesthree complementarity regions (CDRs), designated CDRH1, CDRH2, andCDRH3. The light chain also comprises three CDRs, designated CDRL1,CDRL2, and CDRL3. The amino acid sequences of the CDRs, as well as thenucleotide sequences encoding said amino acid sequences, are shown inTable 1.

TABLE 1 SEQ ID NO: of Nucleotide SEQ ID NO: of CDR's CDR SequenceEncoding CDR Amino Acid Sequence CDRH1 8 9 CDRH2 10 11 CDRH3 12 13 CDRL114 15 CDRL2 16 17 CDRL3 18 19

The presently disclosed inventive concept(s) is also directed to anantibody or antigen-binding fragment comprising a heavy chain variabledomain that comprises an amino acid sequence encoded by SEQ ID NO:4and/or an amino acid sequence as set forth in SEQ ID NO:5, andcompositions comprising same. The presently disclosed inventiveconcept(s) is also directed to an antibody or antigen-binding fragmentcomprising a light chain variable domain that comprises an amino acidsequence encoded by SEQ ID NO:6 and/or an amino acid sequence as setforth in SEQ ID NO:7, and compositions comprising same. The presentlydisclosed inventive concept(s) is further directed to an antibody orantigen-binding fragment comprising a heavy chain that comprises atleast one CDR having an amino acid sequence encoded by at least one ofSEQ ID NOS: 8, 10 and 12 and/or an amino acid sequence as set forth inat least one of SEQ ID NOS: 9, 11 and 13, as well as compositionscomprising same. The presently disclosed inventive concept(s) is furtherdirected to an antibody or antigen-binding fragment comprising a lightchain that comprises at least one CDR having an amino acid sequenceencoded by at least one of SEQ ID NOS: 14, 16 and 18 and/or an aminoacid sequence as set forth in at least one of SEQ ID NOS: 15, 17 and 19,as well as compositions comprising same.

In one particular embodiment, the presently disclosed inventiveconcept(s) is directed to an isolated monoclonal antibody (or antigenbinding fragment thereof) that comprises the light chain variable regionCDR3 (referred to herein interchangeably as “CDRL3”) having the aminoacid sequence of SEQ ID NO:19 and a heavy chain variable region CDR3(referred to herein interchangeably as “CDRH3”) having the amino acidsequence of SEQ ID NO:13. Said antibody specifically binds to an epitopewithin the LRP6 extracellular domain, wherein the LRP6 extracellulardomain has the amino acid sequence of SEQ ID NO:2. The antibody mayfurther include the light chain variable region CDR1 and CDR2 (referredto herein interchangeably as “CDRL1” and “CDRL2,” respectively) havingamino acid sequences of SEQ ID NOS:15 and 17, respectively, and may alsofurther include the heavy chain variable region CDR1 and CDR2 (referredto herein interchangeably as “CDRH1” and “CDRH2,” respectively) havingamino acid sequences of SEQ ID NOS:9 and 11. In addition, the antibodymay possess a heavy chain variable region having an amino acid sequencethat is at least 90% identical to SEQ ID NO:5 and a light chain variableregion having an amino acid sequence that is at least 90% identical toSEQ ID NO:7.

Though in some embodiments the antibodies and antigen binding fragmentsthereof include one or more CDRs that have amino acid sequencesidentical to the corresponding CDR of anti-LRP6-1, in some embodimentscertain CDRs of the antibodies or antigen binding fragments thereof haveamino acid sequences that are substantially similar, but not identicalto the corresponding CDR of anti-LRP6-1. In some embodiments, theantibodies and antigen binding fragments thereof have CDR sequencesidentical to the corresponding CDR sequences of anti-LRP61, except formutations in 6, 5, 4, 3, 2, or 1 CDR amino acid. In some embodiments,the amino acid mutations are present only in CRDL1, CDRL2, CDRH1, orCDRH2. In some embodiments, the mutations are only present in CDRL1,CDRL2, or CDRH1. In some embodiments, the mutations are conservativesequence modifications.

In certain embodiments, the antibodies and antibody fragments have aheavy chain variable region that includes CDR1, CDR2, and CDR3 sequencesand a light chain variable region that includes CDR1, CDR2, and CDR3sequences, where the light chain variable region CDR3 has an amino acidsequence of SEQ ID NO: 19 or conservative modifications thereof, and theheavy chain variable region CDR3 has an amino acid sequence of SEQ IDNO: 13 or conservative modifications thereof. In some embodiments, theheavy chain variable region CDR2 has an amino acid sequence of SEQ IDNO: 11 or conservative modifications thereof. In some embodiments, thelight chain variable region CDR2 has an amino acid sequence of SEQ IDNO: 17 or conservative modifications thereof. In some embodiments, theheavy chain CDR1 has an amino acid sequence of SEQ ID NO: 9 orconservative modifications thereof. In some embodiments the light chainvariable region has an amino acid sequence of SEQ ID NO: 15 orconservative modifications thereof.

In certain embodiments, the antibodies or antigen binding fragmentsthereof have a heavy chain variable region having an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 5. In certain embodiments the antibodies orantigen binding fragments have a light chain variable region having anamino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% identical to SEQ ID NO: 7.

In some embodiments, the antibodies or antigen binding fragments thereofhave a heavy chain variable region CDR3 having an amino acid sequence ofSEQ ID NO: 13 and a heavy chain variable region CDR2 having an aminoacid sequence of SEQ ID NO: 11. In some embodiments, the antibodies orantigen binding fragments thereof also have a heavy chain variableregion CDR1 having an amino acid sequence of SEQ ID NO: 9. In someembodiments, the antigen binding antibody fragments do not include alight chain. For example, in some embodiments the antibody bindingfragments are single domain antibodies, such as but not limited to,NANOBODIES®.

In some embodiments, the antibodies or antigen binding fragments thereofhave a light chain variable region CDR3 having an amino acid sequence ofSEQ ID NO: 19 and a light chain variable region CDR2 having an aminoacid sequence of SEQ ID NO: 17. In some embodiments, the antibodies orantigen binding fragments thereof also have a light chain variableregion CDR1 having an amino acid sequence of SEQ ID NO: 15. In someembodiments, the antigen binding antibody fragments do not include aheavy chain.

The antibodies or antigen binding fragments thereof can be preparedusing an antibody having the V_(H) and/or V_(L) sequences of anti-LRP6-1as starting material to engineer a modified antibody, which modifiedantibody may have altered properties from anti-LRP6-1, but retain theepitope specificity of anti-LRP6-1. An antibody or antigen bindingfragment thereof can be engineered by modifying one or more residueswithin one or both variable regions (i.e., the heavy chain variableregion or the light chain variable region), for example, within one ormore CDR regions and/or within one or more framework regions.Additionally or alternatively, an antibody can be engineered bymodifying residues within the constant region(s), for example, to alterthe effector function(s) and/or immunogenicity of the antibody.

The presently disclosed inventive concept(s) further includes chimericantibodies that comprise at least a portion of a variable region ofAnti-LRP6-1 (i.e., the monoclonal antibody produce by hybridoma HLS2F1,ATCC accession number PTA-10663) and a constant region of an antibodyderived from a human. Such antibodies retain anti-LRP6-1's antigenspecificity and ability to inhibit the Wnt signaling pathway but havereduced immunogenicity in humans compared to anti-LRP6-1.

In some embodiments, the antibodies and antibody fragments are“humanized.” Thus, in some embodiments, the antibodies or antigenbinding fragments thereof are humanized forms of antibodies or fragmentsthereof (such as Fv, Fab, Fab′, F(ab′)2, or other antigen-bindingsubsequences of antibodies) that are principally comprised of thesequence of a human antibody or antibody fragment but contain one ormore CDR from anti-LRP6-1.

Such humanized antibodies or antibody fragments can be generated bysubstituting one or more CDRs of anti-LRP6-1 for the correspondingsequences of a human antibody or antibody fragment. In certainembodiments, a humanized antibody is constructed by replacing 1, 2, 3,4, 5, or 6 of the CDRs of a human antibody with CDRs from anti-LRP6-1.In certain embodiments, a humanized antibody or antibody fragmentcomprises variable regions in which all of the CDRs correspond to CDRsof anti-LRP6-1 and all of the framework regions (FRs) correspond to FRsof a human antibody. In some embodiments, the humanized antibody orantibody fragment has a CDRL3 and CDRH3 of anti-LRP6-1, but retainshuman sequences for one or more of CDRL1, CDRL2, CDRH1, or CDRH2. Insome embodiments the human CDR sequences are selected to be similar insequence to the corresponding anti-LRP6-1 CDR. In such embodiments, thehuman CDRs may have 5, 4, 3, 2, or 1 mutation, either collectively orindividually, compared to their corresponding anti-LRP6-1 CDR. In someembodiments, the mutations are conservative sequence modifications. Insome embodiments, a humanized antibody further comprises a constantregion (Fc) of a human antibody.

In some instances, certain Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues. In someembodiments, humanized antibodies can also comprise residues which arefound neither in the recipient antibody nor in the imported CDR orframework sequences. Methods of making and/or using humanized monoclonalantibodies can be found, for example, in, Sandborn et al.,Gatroenterology, 120:1330 (2001); Mihara et al., Clin. Immunol., 98:319(2001); Yenari et al., Neurol. Res., 23:72 (2001); Morales et al., Nucl.Med. Biol., 27:199 (2000); Richards et al., Cancer Res., 59:2096 (1999);Yenari et al., Exp. Neurol., 153:223 (1998); and Shinkura et al.,Anticancer Res., 18:1217 (1998), all of which are expressly incorporatedin their entirety by reference.

In some embodiments, the hinge region of CH1 of the antibodies ismodified such that the number of cysteine residues in the hinge regionis altered, e.g., increased or decreased. This approach is describedfurther in U.S. Pat. No. 5,677,425 by Bodmer et al., incorporated byreference in its entirety. The number of cysteine residues in the hingeregion of CH1 is altered to, for example, facilitate assembly of thelight and heavy chains or to increase or decrease the stability of theantibody.

In some embodiments, the glycosylation of the antibody or antigenbinding fragment thereof is modified. For example, an aglycoslatedantibody can be made (i.e., the antibody lacks glycosylation).Glycosylation can be altered to, for example, increase the affinity ofthe antibody or antibody fragment for an antigen. Such carbohydratemodifications can be accomplished by, for example, altering one or moresites of glycosylation within the antibody sequence. For example, one ormore amino acid substitutions can be made that result in elimination ofone or more variable region framework glycosylation sites to therebyeliminate glycosylation at that site. Such aglycosylation may increasethe affinity of the antibody for antigen. Such an approach is describedin further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 by Co etal., incorporated by reference in their entirety.

In some embodiments the antibody or antigen binding fragment is madesuch that it has an altered type of glycosylation, such as ahypofucosylated antibody having reduced amounts of fucosyl residues oran antibody having increased bisecting GlcNac structures. Such alteredglycosylation patterns have been demonstrated to increase the ADCCability of antibodies. Such carbohydrate modifications can beaccomplished by, for example, expressing the antibody in a host cellwith altered glycosylation machinery. Cells with altered glycosylationmachinery have been described in the art and can be used as host cellsin which to express recombinant antibodies of the presently disclosedinventive concept(s) to thereby produce an antibody with alteredglycosylation. For example, EP 1,176,195 by Hanai et al. describes acell line with a functionally disrupted FUT8 gene, which encodes afucosyl transferase, such that antibodies expressed in such a cell lineexhibit hypofucosylation. PCT Publication WO 03/035835 by Prestadescribes a variant CHO cell line, Lec13 cells, with reduced ability toattach frucose to Asn(297)-linked carbohydrates, also resulting inhypofucosylation of antibodies expressed in that host cell (see alsoShields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740). PCTPublication WO 99/54342 by Umana et al. describes cell lines engineeredto express glycoprotein-modifying glycosyl transferases (e.g.,beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al. (1999) Nat. Biotech. 17:176-180).

In some embodiments, the PEGylation of the antibodies or antigen bindingfragments thereof is modified. An antibody can be PEGylated, forexample, to increase the biological (e.g., serum) half-life of theantibody. To PEGylate an antibody, the antibody, or fragment thereof,typically is reacted with polyethylene glycol (PEG), such as a reactiveester or aldehyde derivative of PEG, under conditions in which one ormore PEG groups become attached to the antibody or antibody fragment. Incertain non-limiting embodiments, the PEGylation is carried out via anacylation reaction or an alkylation reaction with a reactive PEGmolecule (or an analogous reactive water-soluble polymer). As usedherein, the term “polyethylene glycol” is intended to encompass any ofthe forms of PEG that have been used to derivatize other proteins, suchas mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethyleneglycol-maleimide. In certain embodiments, the antibody to be PEGylatedis an aglycosylated antibody. Methods for PEGylating proteins are knownin the art and can be applied to the antibodies of the presentlydisclosed inventive concept(s). See, for example, EP 0 154 316 byNishimura et al. and EP 0 401 384 by Ishikawa et al.

In some embodiments, the antibodies and antibody fragments have minorvariations in the amino acid sequences of antibodies or antigen bindingfragments thereof described above, providing that the variations in theamino acid sequence maintain at least 75%, such as at least 80%, 90%,95%, or 99%, of the original amino acid sequence, and provided that theantibodies/antibody fragments maintain the ability to specifically bindthe extracellular domain of LRP6. In some embodiments, the modificationsare conservative sequence modifications.

In some embodiments, the monoclonal antibodies of the presentlydisclosed inventive concept(s) neutralize an activity of LRP6.Therefore, said monoclonal antibodies may be referred to as aneutralizing antibody and/or a therapeutic antibody. In one embodiment,the monoclonal antibody specifically inhibits activation of the Wntsignaling pathway. In yet another embodiment, the monoclonal antibodyinhibits the binding of a Wnt ligand to a receptor of the Wnt signalingpathway, thereby preventing activation of the Wnt signaling pathway byWnt ligands. In another embodiment, the monoclonal antibody inhibitsphosphorylation of LRP6. In certain embodiments the antibodies andantibody fragments inhibit the binding of both Wnt1 and Wnt3a to LRP6.In a further embodiment, the monoclonal antibody specifically blocks theover-expression of at least one pro-inflammatory factor and/or at leastone pro-angiogenic factor induced by diabetic conditions. Said factorsmay include but is not limited to, VEGF, ICAM-1, TNF-α, CTGF, andcombinations thereof.

The presently disclosed inventive concept(s) also includes apharmaceutical composition comprising a therapeutically effective amountof at least one of the monoclonal antibodies or antigen fragmentsthereof described herein and compositions comprising same in combinationwith a pharmaceutically acceptable carrier. As used herein, a“pharmaceutically acceptable carrier” is a pharmaceutically acceptablesolvent, suspending agent or vehicle for delivering the compounds of thepresently disclosed inventive concept(s) to the human or animal. Thecarrier may be liquid or solid and is selected with the planned mannerof administration in mind. Examples of pharmaceutically acceptablecarriers that may be utilized in accordance with the presently disclosedinventive concept(s) include, but are not limited to, PEG, liposomes,ethanol, DMSO, aqueous buffers, oils, and combinations thereof.

The compositions of the presently disclosed inventive concept(s)(including but not limited to the pharmaceutical compositions describedimmediately herein above) may further comprise a second agent that has asynergistic effect with the monoclonal antibody, such as but not limitedto, an anti-angiogenic agent, an anti-VEGF reagent, VEGF Trap, AVASTIN®,and the like.

The presently disclosed inventive concept(s) also includes methods ofproducing the monoclonal antibodies (or antigen binding fragmentthereof) described herein above. The monoclonal antibodies and antibodyfragments described herein may be produced by any appropriatemethodology known in the art. For example, preparation of monoclonalantibodies can begin with the production of polyclonal antibodiesgenerated by immunizing a suitable subject (e.g. a mouse) with apolypeptide immunogen (e.g., a polypeptide that includes a portion ofthe LRP6 extracellular domain). At an appropriate time afterimmunization, e.g., when the antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies using standard techniques, such as thehybridoma technique originally described by Kohler and Milstein (1975)Nature 256:495-497) (see also Brown et al. (1981) J. Immunol.127:539-46; Brown et al. (1980) J. Biol. Chem. 255:4980-83; Yeh et al.(1976) Proc. Natl. Acad. Sci. 76:2927-31; and Yeh et al. (1982) Int. J.Cancer 29:269-75), the more recent human B cell hybridoma technique(Kozbor et al. (1983) Immunol. Today 4:72), the EBV-hybridoma technique(Cole et al. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96), or trioma techniques.

The technology for producing monoclonal antibody hybridomas is wellknown (see generally Kenneth, R. H. in Monoclonal Antibodies: A NewDimension In Biological Analyses, Plenum Publishing Corp., New York,N.Y. (1980); Lerner, E. A. (1981) Yale J. Biol. Med. 54:387 402; Gefter,M. L. et al. (1977) Somatic Cell Genet. 3:231 36). Briefly, an immortalcell line (typically a myeloma) is fused to lymphocytes (typicallysplenocytes) from a mammal immunized with an immunogen as describedabove, and the culture supernatants of the resulting hybridoma cells arescreened to identify a hybridoma producing a monoclonal antibody thatbinds to the polypeptide antigen, preferably specifically.

Once the antibodies have been obtained, for example, once individual Bcells have been identified and/or monoclonal antibodies have beenproduced, the sequences encoding the variable regions of theseantibodies can be obtained. The variable region sequences can, forexample, be obtained by first sequencing the antibody protein producedby the hybridoma, B-cell or phage and determining the encoding nucleicacid sequence. In one embodiment, the immunoglobulin variable region (VHand VL) DNA or cDNA may be sequenced instead. Where the antibody isderived from a hybridoma cell line or isolated B-cell, the cDNAsencoding the variable regions may be amplified using PCR by, forexample, the methods described in Babcook et al. (Proc. Natl. Acad. Sci.USA, 93:7843-7848 (1996)), and in PCT Publication No. WO 92/02551. Thecontents of both references are expressly incorporated herein byreference in their entirety.

Thus, the antibodies and antigen binding fragments described herein canbe generated using a method of producing a monoclonal antibody orantigen binding fragment thereof that includes the steps of providing acell that produces a monoclonal antibody or antigen binding fragmentthereof described herein and culturing the cell under conditions thatpermit production of the monoclonal antibody or antigen binding fragmentthereof. In some embodiments, the cell is the hybridoma having ATCCDesignation No. PTA-10663.

As an alternative to preparing monoclonal antibody-secreting hybridomas,a monoclonal specific for LRP6 can be identified and isolated byscreening a recombinant combinatorial immunoglobulin library (e.g., anantibody phage display library or an antibody yeast display library)with the appropriate polypeptide to thereby isolate immunoglobulinlibrary members that bind the polypeptide (see, e.g., Clackson et al.,Nature, 352: 624-628 (1991); and Marks et al., J. Mol. Biol.,222:581-597 (1991), each of which is incorporated by reference).

Additionally, using antibody and antigen binding fragment sequencesprovided herein and known in the art, the monoclonal antibodies andantigen binding fragments, including chimeric or humanized monoclonalantibodies, can be made using standard recombinant DNA techniques. Suchmonoclonal antibodies and antibody fragments can be produced, forexample, using methods described in U.S. Pat. No. 4,816,567; U.S. Pat.No. 5,565,332; Better et al. (1988) Science 240:1041-1043; Liu et al.(1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J.Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci.84:214-218; Nishimura et al. (1987) Cancer Res. 47:999-1005; Wood et al.(1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al.(1986) Biotechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al.(1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060, or U.S. Pat. No.5,916,771, each of which is hereby incorporated by reference in itsentirety.

The presently disclosed inventive concept(s) also includes isolatednucleic acid molecules encoding the amino acid sequence of any of themonoclonal antibodies (or fragments thereof) described herein above,including but not limited to, the heavy and/or light chain variabledomains of said monoclonal antibodies as well as one or more CDRs ofsaid heavy/light chain variable domains. In one embodiment, thepresently disclosed inventive concept(s) comprises isolated nucleic acidmolecules encoding at least one of (a) a heavy chain variable regionhaving a CDR1 of SEQ ID NO:9; (b) a heavy chain variable region having aCDR2 of SEQ ID NO: 11; (c) a heavy chain variable region having a CDR3of SEQ ID NO: 13; (d) a light chain variable region having a CDR1 of SEQID NO: 15; (e) a light chain variable region having a CDR2 of SEQ ID NO:17; and (f) a light chain variable region having a CDR3 of SEQ ID NO:19. In another embodiment, the presently disclosed inventive concept(s)comprises isolated nucleic acid molecules comprising at least one of SEQID NOS:4, 6, 8, 10, 12, 14, 16 and 18.

In one particular embodiment, the presently disclosed inventiveconcept(s) comprises an isolated nucleic acid molecule encoding a heavychain variable region having a CDR3 of SEQ ID NO:13. The heavy chainvariable region of said nucleic acid molecule may further include a CDR1of SEQ ID NO:9 and a CDR2 of SEQ ID NO:11.

In another particular embodiment, the presently disclosed inventiveconcept(s) comprises an isolated nucleic acid molecule encoding a lightchain variable region having a CDR3 of SEQ ID NO:19. The light chainvariable region of said nucleic acid molecule may further include a CDR1of SEQ ID NO:15 and a CDR2 of SEQ ID NO:17.

Nucleic acids of the presently disclosed inventive concept(s) can beobtained using standard molecular biology techniques. For antibodiesexpressed by hybridomas (e.g., hybridomas prepared as described above),cDNAs encoding the light and heavy chains of the antibody made by thehybridoma can be obtained using standard PCR amplification or cDNAcloning techniques. For antibodies obtained from an immunoglobulin genelibrary (e.g., using phage display techniques), nucleic acid encodingthe antibody can be recovered from the library.

In some embodiments the isolated nucleic acid molecules have at least80%, 85%, 90%, 95%, or 100% sequence identity to the nucleic acidmolecules described above.

The presently disclosed inventive concept(s) also comprises a vectorcomprising any of the isolated nucleic acid molecules described hereinabove. The presently disclosed inventive concept(s) further include ahost cell comprising said nucleic acid molecule(s) and/or said vector.

The presently disclosed inventive concept(s) also comprises a cell orcell line expressing the monoclonal antibodies described herein above,including but not limited to, Anti-LRP6-1. In one embodiment, the cellline is a hybridoma cell line. In yet another embodiment, the cell lineis the hybridoma cell line deposited as described herein above.

The monoclonal antibodies, antibody fragments and nucleic acidcompositions described herein have numerous therapeutic utilities forthe treatment of Wnt pathway associated disorders or disorders involvingLRP6 activity. These molecules and/or compositions can be administeredto subjects, including human subjects, to treat or prevent a variety ofdisorders, including but not limited to, inflammation, vascular leakage,fibrosis, abnormal neovascularization, and cancer.

The presently disclosed inventive concept(s) is further related to amethod of inhibiting activation of the Wnt signaling pathway, saidmethod comprising administering a composition comprising any of themonoclonal antibodies described in detail herein above. If thecomposition does not include a second agent, the method may furthercomprise the administration of a second agent that has a synergisticeffect with the monoclonal antibody, such as but not limited to, ananti-angiogenic agent, an anti-VEGF reagent, VEGF Trap, AVASTIN®, andthe like.

The presently disclosed inventive concept(s) is further related to amethod of inhibiting enzyme activity and/or enzyme production of atleast one angiogenic, inflammatory and/or fibrogenic factor of DR. Saidfactors include but are not limited to, VEGF, ICAM-1, TNF-α, and CTGF.Said method comprises administering to a subject suffering from orpredisposed to DR any of the pharmaceutical compositions described indetail herein above. If the composition does not include a second agent,the method may further comprise the administration of a second agentthat has a synergistic effect with the monoclonal antibody, such as butnot limited to, an anti-angiogenic agent, an anti-VEGF reagent, VEGFTrap, AVASTIN®, and the like.

The presently disclosed inventive concept(s) is also directed to amethod of mediating/attenuating at least one retinal condition selectedfrom the group consisting of retinal leukostasis, inflammation, vascularleakage, fibrosis, abnormal neovascularization (such as but not limitedto, retinal neovascularization and/or choroidal neovascularization), andcarcinogenesis in the retina. Said method comprises administering any ofthe pharmaceutical compositions described in detail herein above. If thecomposition does not include a second agent, the method may furthercomprise the administration of a second agent that has a synergisticeffect with the monoclonal antibody, such as but not limited to, ananti-angiogenic agent, an anti-VEGF reagent, VEGF Trap, AVASTIN®, andthe like.

The presently disclosed inventive concept(s) is further directed to amethod of inhibiting and/or decreasing the occurrence and/or severity ofat least one condition selected from the group consisting of diabeticretinopathy, diabetic macular edema, macular degeneration (including butnot limited to, age related macular degeneration), cancer, and otherinflammatory and neovascular disorders of the eye. Said method comprisesproviding a subject suffering from or predisposed to at least one of theabove conditions, and administering an effective amount of any of thepharmaceutical compositions described in detail herein above, wherebythe pharmaceutical composition inhibits activation of the Wnt signalingpathway, thereby inhibiting and/or decreasing the occurrence and/orseverity of said condition/disorder. If the composition administered tothe subject does not contain a second agent, the method may furthercomprise the administration of a second agent that has a synergisticeffect with the monoclonal antibody, such as but not limited to, ananti-angiogenic agent, an anti-VEGF reagent, VEGF Trap, AVASTIN®, andthe like.

In any of the methods described herein above, the method ofadministration may comprise injection of the composition into thevitreous of the eye.

Examples are provided hereinbelow. However, the present inventiveconcept(s) is to be understood to not be limited in its application tothe specific experimentation, results and laboratory procedures. Rather,the Examples are simply provided as one of various embodiments and aremeant to be exemplary, not exhaustive.

EXAMPLE 1

Diabetic retinopathy (DR), the leading cause of blindness in the workingage population, represents a common concern in types 1 and 2 of diabetesmellitus (DM). Accumulating evidence suggests that DR is a chronicinflammatory disorder. Retinal inflammation is believed to play acausative role in vascular leakage, which can lead to diabetic macularedema, and in retinal neovascularization (NV). It has been shown thatlevels of soluble intercellular adhesion molecule-1 (ICAM-1) andvascular cell adhesion molecule-1 are significantly higher in thevitreous from patients with proliferative diabetic retinopathy than innon-diabetic vitreous. Increased ICAM-1, vascular cell adhesionmolecule-1, and e-selectin levels were found in the serum from patientswith diabetic microangiopathy. In diabetic animal models, increasedretinal ICAM-1 expression is believed to be responsible for leukocyteadhesion or leukostasis and increased vascular permeability. Leukostasisis believed to contribute to capillary nonperfusion and local ischemia,which subsequently induces the overexpression of vascular endothelialgrowth factor (VEGF). Increased VEGF levels are responsible for theretinal vascular leakage and retinal NV. Recent studies have indicatedthat oxidative stress, induced by hyperglycemia, contributes to retinalinflammation in diabetes. However, the pathogenic mechanisms by whichdiabetes and oxidative stress induce inflammation are not certain at thepresent time.

Wnts are a group of secreted, cysteine-rich glycoproteins, which bind toa coreceptor complex of frizzled (Fz) receptors and low-densitylipoprotein receptor-related protein 5 or 6 (LRP5/6) and regulateexpression of a number of target genes through an intracellularsignaling pathway, namely the Wnt pathway. In the absence of Wntligands, β-catenin, a down-stream effector of the canonical Wnt pathway,is phosphorylated by a protein complex containing glycogen synthasekinase-3β. The phosphorylated β-catenin is constantly degraded, toprevent its accumulation. On binding of certain Wnts to the Fz-LRP5/6coreceptors, phosphorylation of β-catenin is inhibited, which preventsthe degradation of β-catenin and results in its accumulation. β-cateninis then translocated into the nucleus, associates with T-cell factor forDNA binding, and regulates expression of target genes including VEGF.

LRP5/6 are known to play a critical role in Wnt/β-catenin signaling. Onbinding with Wnt ligands, LRP6 dimerizes with Fz receptor, which is thefirst and essential step in activation of the Wnt pathway. Thecytoplasmic domain of LRP6 has multiple modular phosphorylation sites,and phosphorylation of LRP6 is an essential event for activation of thecanonical Wnt pathway, as the phosphorylation of LRPE6 promotes therecruitment of the scaffold protein Axin, and thus, activates thecanonical Wnt signaling pathway.

Recent evidence indicates that the canonical Wnt pathway plays a role inangiogenesis. Extensive studies have shown that the Wnt pathwayup-regulates nuclear factor κB, signal transducer and activator oftranscription 3 and a number of inflammatory factors, and thus, plays akey role in inflammation. The present Example investigated the possiblerole of the Wnt signaling pathway in DR by using human donor eyes,diabetic animal models, and cultured cells.

Materials and Methods of Example 1

Human Tissue: Normal and diabetic eyes fixed in 10% neutral bufferedformalin (NBF) within 12 hours postmortem and were obtained fromNational Diseases Research Interchange (Philadelphia, Pa.) with fullethical approval for use in research. Diabetic eyes were categorizedaccording to a standardized protocol (Khaliq et al., Lab Invest,78:109-116 (1998)).

Animals: Akita mice were purchased from the Jackson Laboratory (BarHarbor, Me.), and Brown Norway rats were purchased from Charles River(Wilmington, Mass.). Care, use, and treatment of all animals in thisstudy were in strict agreement with the Statement for the Use of Animalsin Ophthalmic and Vision Research from the Association for Research inVision and Ophthalmology.

Isolation and Culture of Bovine Retinal Capillary Endothelial Cells andPericytes: Bovine retinal capillary endothelial cells (RCEC) andpericytes were isolated from bovine eyes, as described by Grant and Guay(Invest Opthalmol Vis Sci, 32:53-64 (1991)) with some modifications. Atpassage 3 or 4, the purity of the cells in culture was determined. Theidentity of RCEC was confirmed by a characteristic cobblestonemorphology and the incorporation of acetylated low-density lipoproteinlabeled with a fluorescent probe, Dil(1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate)(Biomedical Technologies, Inc.; Stoughton, Mass.). Purity of thepericyte culture was determined by immunostaining using a fluoresceinisothiocyanate-conjugated antibody specific to α-smooth muscle actin(Sigma; St. Louis, Mo.).

Induction of Diabetes in Rats: Experimental diabetes was induced by anintraperitoneal injection of streptozotocin (STZ) (50 mg/kg in 10 mmol/Lof citrate buffer; pH 4.5) into anesthetized Brown Norway rats (8 weeksof age) after an overnight fast. Age-matched control rats received aninjection of citrate buffer alone for non-diabetic control. Bloodglucose levels were measured 48 hours after the STZ injection andmonitored weekly thereafter. Only the animals with glucose levels >350mg/dl were considered diabetic.

The Oxygen-Induced Retinopathy Model and Analysis of Retinal NV: Theoxygen-induced retinopathy (OIR) model was induced in Brown Norway ratsas described previously (Ricci., Doc Opthalmol, 74:171-177 (1990)).Quantification of preretinal vascular cells was described by Smith etal. (Invest Opthalmol Vis Sci, 35:101-111 (1994)). Briefly, the eyes ofeight rats from each group at postnatal day 18 (P18) were enucleated,fixed with 10% formaldehyde, sectioned, and then stained with H&E. Thenuclei of vascular cells on the vitreal side of the retina were countedunder a light microscope in a double-blind study. Ten sagittal sectionsfrom each eye were examined, and cell numbers were averaged in eachgroup of animals. The average number of preretinal vascular nuclei wascompared with that in the control group by using Student's t-test.

Retinal Angiography with High-Molecular-Weight Fluorescein: Rats at P18were anesthetized with 10 mg/kg xylazine plus 75 mg/kg ketamine i.p. andperfused with 50 mg/ml high molecular weight fluoresceinisothiocyanate-dextran (molecular weight 2×10⁶; Sigma) viaintraventricle injection as described by Smith et al. (Invest OpthalmolVis Sci, 35:101-111 (1994)). The animals were immediately euthanized.The eyes were enucleated and fixed with 4% paraformaldehyde in PBS for10 minutes. The retina was then separated from the eyecup and fixed with4% paraformaldehyde for 3 hours. Several incisions were made to theretina, which was flat-mounted on a gelatin-coated slide. Thevasculature was then examined under a fluorescence microscope (Axioplan2Imaging; Carl Zeiss; Jena, Germany).

Immunohistochemistry: Immunohistochemistry was performed as described(Chen et al., Invest Opthalmol Vis Sci, 47:1177-1184 (2006)). Theprimary antibodies specific for LRP5/6 (Abcam; Cambridge, Mass.) andhypoxia-inducible factor-1α (HIF-1α) (Santa Cruz Biotechnology; SantaCruz, Calif.) were used at a dilution of 1:200, and antibody forβ-catenin (Cell Signaling Technology, Danvers, Mass.) at a dilution of1:300. The secondary antibodies were fluoresceinisothiocyanate-conjugated goat anti-mouse IgG (Jackson ImmunoResearchLaboratory, Inc; West Grove, Pa.) and Texas Red-conjugated goatanti-rabbit IgG (Jackson ImmunoResearch Laboratory) at a dilution of1:200.

Measurement of Reactive Oxygen Species Generation: Cellular oxidativestress was determined by measuring intracellular reactive oxygen species(ROS) generation (Degli, Methods, 26:335-340 (2002); and Amer et al.,Eur J Haematol, 70:84-90 (2003)). Briefly, the treated and untreatedcells at a density of 2×10⁶ cells/ml were incubated with freshlyprepared 5-(and 6-)chloromethyl-2′, 7′-dichlorodihydrofluoresceindiacetate, acetyl ester (CM-H2-DCF-DA) at 37° C. in the dark. TheCM-H2-DCF-DA-loaded cells were rinsed twice in PBS and analyzedimmediately by fluorometer at 488 nm excitation and 530 nm emission.Data were expressed as fluorescence intensity in arbitrary units fromthe average of three separate experiments.

Intravitreal Injection of Dickkopf Homolog 1: Briefly, animals wereanesthetized with a 50:50 mix of ketamine (100 mg/ml) and xylazine (20mg/ml), and their pupils were dilated with topical application ofphenylephrine (2.5%) and tropicamide (1%). A sclerotomy was createdapproximately 0.5 mm posterior to the limbus with a blade, and a glassinjector (˜33 gauge) connected to a syringe filled with 3 μl of thespecified concentrations of Dickkopf homolog 1 (DKK1) or bovine serumalbumin (BSA) was introduced through the sclerotomy into the vitreouscavity.

Soluble ICAM-1 Enzyme-Linked Immunosorbent Assay: A commercial solubleICAM-1 (sICAM-1) enzyme-linked immunosorbent assay kit (R&D Systems,Inc.; Minneapolis, Minn.) was used to measure sICAM-1 levels in mouseretinal tissues, following the manufacturer's protocol. The samples frommouse tissues were diluted 10 times to ensure that the sICAM-1concentration fell within the range of the sICAM-1 standard curves.

Western Blot Analysis: The same amounts of retinal proteins from eachrat or mouse were used for Western blot analysis using specific primaryantibodies for each protein and blotted with a horseradishperoxidase-conjugated secondary antibody (Chen et al., Invest OpthalmolVis Sci, 47:1177-1184 (2006)). The signal was developed with achemiluminescence detection kit (ECL; Amersham International;Piscataway, N.J.). Blots were then stripped and reblotted with anantibody specific for β-actin.

The primary antibodies used are specific for LRP5/6 (Santa CruzBiotechnology) and HIF-1α (Santa Cruz Biotechnology) at a dilution of1:500, and the anti-β-catenin antibody (Cell Signaling Technology) at adilution of 1:3000.

Vascular Permeability Assay: Vascular permeability was quantified byusing Evans blue as a tracer as described previously (Gao et al.,Diabetologica, 46:689-698 (2003)).

Results of Example 1

The Wnt Pathway Is Activated in the Retina of Human Patients with DR: Toevaluate the activation status of the Wnt pathway in the retina ofdiabetic patients, retinal levels of total β-catenin, an essentialeffector of the canonical Wnt pathway, were measured. Ocular sectionsfrom six diabetic donors with nonproliferative diabetic retinopathy(NPDR) and those from five non-diabetic donors were stained forβ-catenin by using immunohistochemistry. Under the same developmentintensity, there was a statistically significant increase in β-cateninstaining intensity in the inner retina from the donors with NPDR, whilethere were only basal levels of β-catenin signal in the retina from thenon-diabetic donors (FIG. 1, A-E). Moreover, immunohistochemistry showedthat the donors with NPDR had more intensive β-catenin signals (browncolor) in the nuclei of the retinal cells, compared with that in thenon-diabetic donors (FIG. 1), indicating increased nuclear translocationof β-catenin in the retinas from patients with NPDR.

Activation of the Wnt Pathway in the Retinas of Akita Mice, STZ-InducedDiabetic Rats, and Rats with OIR: To confirm the activation of the Wntpathway in the retina of DR animal models, β-catenin levels weremeasured in the retinas from Akita mice, a genetic model of type-1diabetes, STZ-induced diabetic rats, and in OIR rats, a model ofischemia-induced retinal NV. As shown by Western blot analysis,β-catenin levels were elevated in the retinas from Akita mice at the ageof 16 weeks, compared with that in their non-diabetic littermates (FIG.2A). Similarly, retinal β-catenin levels were also elevated inSTZ-diabetic rats at 16 weeks after the induction of diabetes, comparedwith age-matched non-diabetic rats (FIG. 2B). In rats at the age ofpostnatal day 16 (P16) under constant normoxia, low levels of β-cateninwere detected in the retina, while the OIR rats at the same age showeddramatically increased β-catenin levels in the retina (FIG. 2C).

To identify the cellular location of the β-catenin accumulation, ocularsections from the eyes of Akita mice, STZ-diabetic rats, OIR rats, andtheir respective controls were stained with an antibody specific forβ-catenin by using immunohistochemistry. More intensive β-cateninsignals (green color) were detected in the inner retinas of the Akitamice, STZ-diabetic rats, and OIR rats, when compared with theirrespective controls (FIG. 2, D-Q). Increased β-catenin signals in thenuclei of retinal cells were also observed in the diabetic animals andOIR rats (FIG. 2, D-Q).

Increased Retinal Levels of LRP5/6 in Diabetic and OIR Rats: To furtherassess the activation status of the Wnt pathway, retinal levels ofLRP5/6, coreceptors in the Wnt pathway, were measured by Western blotanalysis. The results showed that retinal levels of LRP6 were higher inthe retinas from STZ-induced diabetic rats at 16 weeks following theonset of diabetes than that in non-diabetic controls (FIG. 3A).Similarly, retinal LRP6 levels were also elevated in the retinas fromOIR rats at the age of P16, compared with age-matched normoxia controls(FIG. 3B).

Immunohistochemical analysis in ocular sections showed increased LRP5/6signals in the inner retina of STZ-induced diabetic rats (green color inFIGS. 3, C and D), compared with non-diabetic controls. In OIR rats, themore intensive LRP5/6 signals were detected primarily in the retinalvasculature (FIGS. 3, E and F).

Hypoxia and Oxidative Stress Are Responsible for the Wnt PathwayActivation in Diabetes: To identify the cause for the Wnt pathwayactivation in diabetes, the effects of hypoxia and hyperglycemia, knownpathogenic factors of DR, on Wnt signaling were evaluated in vitro. Asshown by Western blot analysis, exposure of primary RCEC to hypoxia (2%oxygen) for 14 hours increased the total β-catenin levels (FIG. 4A),indicating that hypoxia is a causative factor for the Wnt pathwayactivation in the retina of the diabetic and OIR models.

RCEC were also exposed to 30 mmol/L glucose for 24 hours, in thepresence and absence of 10 μmol/L aminoguanidine, which is known to haveanti-oxidant activities. The subcellular distribution of β-catenin inRCEC was determined by using immunocytochemistry. In the cells culturedunder the low glucose medium (5 mmol/L glucose and 25 mmol/L mannitol),β-catenin was distributed primarily in the cytosol and membrane, and wasundetectable in the nuclei (green color in FIG. 4B). The high glucosemedium induced β-catenin nuclear translocation (FIG. 4C), indicatingthat high glucose alone is sufficient to activate the Wnt pathway. Underthe same condition, aminoguanidine inhibited the nuclear translocationof β-catenin induced by high glucose (FIG. 4D). Consistently, Westernblot analysis using isolated nuclear proteins showed that nuclear levelsof β-catenin were elevated in the RCEC exposed to the high glucosemedium, compared with those in the low glucose medium. Aminoguanidineblocked the high glucose-induced increase of nuclear β-catenin levels,suggesting that oxidative stress is responsible for the highglucose-induced activation of the Wnt pathway (FIG. 4E).

Blockade of the Wnt Pathway Ameliorates Retinal Inflammation, VascularLeakage, and NV in DR Models: To further establish the causative role ofthe Wnt pathway activation in DR, the Wnt pathway activation was blockedin the retinas of the DR models by using DKK1, a specific inhibitor ofthe Wnt pathway. An intravitreal injection of different doses ofpurified DKK1 into STZ-diabetic rats reduced retinal soluble ICAM-1levels in a dose-dependent manner, when compared with that in thecontralateral eyes injected with the same amounts of BSA, indicatingthat Wnt signaling is responsible for retinal inflammation in diabeticrats (FIG. 5A). To evaluate the role of Wnt signaling in retinalvascular leakage in diabetic rats, purified DKK1 was injected into thevitreous of the right eye (1.2 μg/eye) of STZ-diabetic rats at 16 weeksfollowing the onset of diabetes, and the same amounts of BSA into thecontralateral eyes for control. Retinal vascular leakage was measured 48hours after the injection by using Evans blue as a tracer, andnormalized by total retinal protein concentrations. Consistently,vascular permeability assays showed that retinal vascular leakage wassignificantly decreased in the eyes injected with DKK1 in diabetic rats,compared with that in the contralateral eyes injected with the same doseof BSA (FIG. 5B).

The Wnt pathway was also blocked by injection of DKK1 (1.0 μg/eye) intoOIR rats at age P14. Two days after the injection, expression ofpro-inflammation factor such as COX2 and permeability factor VEGF wassignificantly down-regulated (FIGS. 5, C and D). Consistently, vascularpermeability assays showed that retinal vascular leakage wassignificantly decreased in the eyes injected with 1 μg/eye DKK1,compared with that in the contralateral eyes injected with the same doseof BSA (FIG. 5E).

To evaluate the role of Wnt signaling in the ischemia-induced retinalNV, DKK1 was injected into the vitreous of OIR rats at the age of P14.The retinal vasculature was visualized by fluorescein angiography inwhole-mounted retina at P18. The DKK1 injection induced apparentdecreases of neovascular areas and tufts, compared with thecontralateral eyes injected with BSA (green color in FIG. 5, F-I).Retinal NV was quantified by counting preretinal vascular cells, whichshowed significant decreases in preretinal vascular cells in theDKK1-injected eyes compared with that in the contralateral eyes injectedwith BSA (FIG. 5J).

Blockade of Wnt Signaling Attenuates the High Glucose-Induced HIF-1Activation and ROS Generation: HIF-1 activation is known to play acrucial role in the overexpression of VEGF and retinal NV in DR. Here itwas examined whether the role of Wnt signaling is through HIF-1.Cultured RCEC were exposed to 30 mmol/L glucose in the presence andabsence of different concentrations of DKK1 for different durations,with 5 mmol/L glucose and 25 mmol/L mannitol as negative controls, and 1μg of tumor necrosis factor (TNF)-α as the positive control. As shown byimmunocytochemistry using an anti-HIF-1α antibody, DKK1 inhibited theHIF-1α nuclear translocation, a key step in its activation, induced bythe high glucose medium (green color in FIG. 6, A-D).

As oxidative stress is believed to be a key pathogenic factor in DR, theeffect of Wnt signaling on ROS generation induced by high glucose andTNF-α was evaluated. As shown by ROS measurement, both TNF-α and thehigh glucose medium (30 mmol/L) significantly increased ROS productionin RCEC, compared with the low glucose medium. DKK1 showed adose-dependent (6.25 to 100 nmol/L) reduction of ROS generation inducedby TNF-α and high glucose. At high concentrations (50 and 100 nmol/L),DKK1 decreased the ROS generation to the same extent as that of 10μmol/L of aminoguanidine (FIG. 6E).

Discussion of Example 1

The Wnt signaling pathway has been shown to regulate multiple biologicaland pathological processes. However, the association of the Wnt pathwaywith DR has not been reported previously. The present Exampledemonstrates for the first time that the Wnt pathway is activated byoxidative stress and hypoxia in DR in humans and animal models.Furthermore, it has been shown herein that blockade of Wnt signalingwith a specific inhibitor of the Wnt pathway ameliorates retinalinflammation, vascular leakage, and NV in the DR models, indicating thatthe Wnt pathway plays a causative role in DR. Therefore, theseobservations have established a new pathogenic role for the Wnt pathway.

β-catenin is an essential down-stream effector in the canonical Wntpathway. The present results obtained using human ocular sectionsrevealed increased retinal levels of β-catenin and enhanced nucleartranslocation, a key step in the activation of β-catenin, in the innerretinal cells in patients with DR, compared with that in non-diabeticdonors. The location of the β-catenin activation in the inner retinacorrelates with the pathological changes in DR. The activation of Wntsignaling in the retina with NPDR, which manifests inflammation andvascular leakage but lacks of NV, indicate that the Wnt activation canoccur at early stages of DR, before the proliferative stages.

To confirm the activation of the Wnt pathway in the retina with DR,retinal β-catenin levels were examined in three animal models of DR.STZ-induced diabetes is a commonly used type-1 diabetic model. Akitamouse is a genetic model of type-1 diabetes. Both of the models havebeen shown to develop retinal inflammation and vascular leakage but notretinal NV, and thus, are NPDR models. Western blot analysis andimmunohistochemistry both showed that total levels of β-catenin werehigher in the retinas of STZ-diabetic rats than in the age-matchednon-diabetic controls. Similarly, Akita mice also had increasedβ-catenin levels compared with their non-diabetic littermates. Theresults from these diabetic models indicate that the activated Wntpathway correlates with retinal inflammation and vascular leakage.

OIR is a commonly used model of ischemia-induced retinal NV. Although itis not a diabetic model, the pathological features of this model, suchas preretinal NV, vascular leakage, and overexpression of HIF-1 and VEGFin the retina, resemble that of proliferative diabetic retinopathy.Thus, OIR is commonly accepted as a proliferative diabetic retinopathymodel. In OIR rats, β-catenin levels were also increased in the innerretina. These results indicate a potential role of the Wnt pathway inischemia-induced retinal NV.

LRP5/6 are closely related coreceptors of Wnt ligands. To confirm theactivation of the Wnt pathway in DR, the retinal levels of LRP5/6 havebeen measured in the DR models. Western blot analysis andimmunohistochemistry both showed that the retinal levels of LRP5/6 wereelevated in the STZ-induced diabetic and OIR models. In contrast, nosignificant changes of the Fz receptor levels were detected in theretina of both of the models. Together with the β-catenin accumulationin these models, these results demonstrate that the Wnt pathway isoveractivated in DR.

DR is a complex and multifactorial disorder. It has been shown thathypoxia and hyperglycemia are the major pathogenic factors. To identifythe cause for the Wnt pathway activation in diabetes, the impacts ofhypoxia and high glucose on Wnt signaling were assessed. In culturedretinal endothelial cells, hypoxia and high glucose medium induced theaccumulation of β-catenin and its nuclear translocation. Theseexperiments indicate that hypoxia and hyperglycemia are causativefactors for the Wnt pathway activation in diabetes.

Oxidative stress induced by hyperglycemia has been shown to be a keypathogenic factor for retinal inflammation and vascular injury. To testthe role of the oxidative stress in the Wnt pathway activation inducedby high glucose, aminoguanidine was used, as it has antioxidantactivities. Both immunocytochemical and Western blot analyses showedthat the β-catenin nuclear translocation induced by high glucose mediumcan be attenuated by aminoguanidine alone, which indicates that theoxidative stress induced by high glucose is a direct cause of the Wntpathway activation in DR.

The Wnt pathway is known to be activated under many pathologicalconditions. To establish the causative role of activated Wnt signalingin DR, the Wnt pathway in the DR models was blocked by using DKK1, aspecific peptide inhibitor of the Wnt pathway. DKK1 is known to bind tocoreceptors LRP5/6 with high specificity and affinity, and block thedimerization of LRP5/6 with the Fz receptor, an essential step in Wntpathway activation. In the DR models, an intravitreal injection of DKK1alone is sufficient to mitigate retinal inflammation as it blocks theoverexpression of pro-inflammatory factors such as ICAM-1 and COX-2.Similarly, DKK1 also reduced retinal vascular leakage and amelioratedthe ischemia-induced retinal NV. These results indicate that blockade ofthe Wnt pathway alone is sufficient to ameliorate DR. Further,activation of the Wnt pathway alone without high glucose in culturedcells was sufficient to induce VEGF expression. These results indicatethat the Wnt pathway activation plays a causative role in DR. Thisconclusion is consistent with previous observations in other tissuesthat the Wnt pathway mediates inflammation and angiogenesis.

To elucidate the mechanism by which Wnt signaling mediates DR, theeffects of the Wnt pathway in oxidative stress were evaluated. Incultured endothelial cells, ROS generation was significantly elevated byhigh glucose and by TNF-α, an inflammatory factor. Blocking Wntsignaling with DKK1 inhibited the ROS generation induced by high glucosemedium and TNF-α. These results indicate that the pathogenic role of theWnt pathway in DR may be via induction of oxidative stress andsubsequently induction of inflammation in the retina.

In summary, this Example provides the first evidence showing that theWnt pathway activation is a novel pathogenic mechanism for DR in bothhuman patients and in animal models. Thus, the Wnt pathway represents anew target for pharmaceutical intervention of DR and therefore hastherapeutic potential in the treatment of DR.

EXAMPLE 2

Retinal NV is a major pathological feature leading to vision loss in DR.VEGF is a well-known key factor in stimulating the retinal NV formationin the DR. Example 1 above demonstrates that the Wnt pathway isup-regulated in the retina of diabetic animals and in anischemia-induced animal model. Furthermore, Example 1 also demonstratesthat inhibition of the Wnt pathway on the top using Dkk1, a Wnt pathwayinhibitor through binding of LRP6, reduces retinal VEGF expression;retinal inflammation and retinal permeability in the retina of STZinduced diabetic model and OIR model.

Despite the above facts that suggest that Dkk1 has great potential inthe treatment of retinal NV in DR, it is noteworthy that the preventiveeffect of Dkk1 in the retina is only temporal due to its shorthalf-life, which requires multiple introvitreous injections and thusserious injury to the eye. Moreover, Dkk1 cannot be expressed as asoluble protein from E. coli, and therefore the cost of its synthesis ishigh. Thus, other inhibitors of Wnt which retain the anti-angiogenicactivities and have a longer half-life is desired. Since the essentialanti-angiogensis activities of Dkk1 are through binding to the LRP6 thusblocking the LRP6 activity, the presently disclosed inventive concept(s)provides an antibody that directly blocks LRP6 activity, therebyinhibiting the Wnt pathway and consequently preventing retinalinflammation and retinal NV. One advantage of this antibody over apeptide inhibitor is that the effect of the antibody lasts longer than 4weeks in vivo. Moreover, since antigen-antibody binding is more specificand higher affinity, this monoclonal Anti-LRP6-1 will have the same ormore potent anti-angiogenic activity on DR than that of Dkk1.

Results of Example 2

Generation of mouse mAbs that neutralize LRP6: A number of clones ofmouse monoclonal antibodies were generated using standard hybridomatechniques using the recombinant ectodomain of LRP6 (described in detailin the other Examples). Out of numerous positive clones of mAb, the mAbAnti-LRP6-1 (the hybridoma producing said antibody deposited with theATCC as described herein above), was identified in this manner.Anti-LRP6-1 was selected based on its specificity for endogenous LRP6 invarious cell lines.

Anti-LRP6 antibodies neutralize human LRP6-mediated cellular activities:

Anti-LRP6 mAb inhibits LRP6 activity: Next, inhibition of LRP6phosphorylation by anti-LRP6 antibodies was measured. Under normalconditions, LRP6 is not activated. Wnt stimulates LRP6 activation byphosphorylation at its PPPSP motif, which is reiterated five times inthe cytoplasmic domain of LRP6; thus, Wnt 3a containing medium was usedfor ligand-mediated receptor activation. The data showed that whenAnti-LRP6-1 was introduced into Wnt3a-containing medium before itsaddition to the cells, LRP6 phosphorylation was inhibited in adose-dependent manner (FIG. 7) with IC₅₀ values in the range of0.21-0.44 μmol/L.

Anti-LRP6-1 mAb inhibits Wnt signaling pathway activation: Since LRP6activation is the key step for Wnt signaling pathways, causing coreeffector β-catenin accumulation, the ability of Anti-LRP6-1 to inhibitWnt signaling downstream from β-catenin accumulation was also assessed.RCEC cells were pretreated with and without Anti-LRP6-1 for 10 minutes,and then separately distributed to three different groups that wereexposed to Wnt3a-containing medium, hypoxia, or high glucose,respectively. The data presented in FIG. 8 demonstrate that Anti-LRP6-1essentially completely inhibited said stimulation.

Anti-LRP6-1 mAb inhibits endothelial cells' proliferation: The Wntsignaling pathway has been implicated in angiogenesis; hence,[¹⁴C]thymidine incorporation into RCEC DNA was measured as a model forinhibition of endothelial cell proliferation and survival. Ascintillation proximity assay was used to measure [¹⁴C]thymidineincorporation into RCECs. RCECs were plated on 96-well scintillantplates, and 48 hours later [¹⁴C]thymidine was added to the cells andmixed with different concentrations of Anti-LRP6-1 or DKK1. RCECs werethen exposed to 1% O₂ for 3 days, and the radioactivity incorporatedinto the cell monolayer was determined and the percentage inhibition ofthe maximal signal was calculated. The results demonstrated thatthymidine incorporation was inhibited by Anti-LRP6-1; the IC₅₀ valuesspan a broad range (0.5-2 μmol/L). However, compared with Dkk1,Anti-LRP6-1 has a more potent inhibitory effect on the proliferation ofendothelial cells (FIG. 9).

Anti-LRP6-1 mAb decreases VEGF over-expression: The biological effectsof Anti-LRP6-1 on VEGF over-expression were also measured. RCECs werepretreated with Anti-LRP6-1 for 10 min, and then exposed to hypoxia for24 hours. The VEGF levels in the culture medium were measured using anELISA kit (R&D Systems, Inc., MN) following the manufacturer's protocol.The results demonstrated that Anti-LRP6-1 significantly decreased VEGFover-expressed in the endothelial cells exposed to hypoxia conditions.Similar results were obtained in several additional experiments (FIG.10).

Anti-LRP6-1 attenuates the generation of reactive oxygen species (ROS)induced by high glucose: RCEC were exposed to 30 mM glucose in theabsence or presence of various concentrations of Anti-LRP6-1 or DKK1(6.25-125 nM) for 2 hours. As shown by the intracellular ROS generationassay, high glucose induced the generation of ROS. Both Anti-LRP6-1 andDKK1 blocked ROS generation. However, Anti-LRP6-1 has a more potentinhibitory effect than that of Dkk1, and in a concentration-dependentmanner (FIG. 11).

Anti-LRP6-1 mAb attenuates retinal vascular leukostasis in the diabeticrats: STZ diabetic rats at 2 weeks after the onset of diabetes receivedan intravitreal injection of 5 μg/eye of Anti-LRP6-1 in the treatmentgroup and the same dose of Control mouse IgG and Dkk1 in the controlgroup. Four weeks after the injection, the animals were perfused toremove circulating leukocytes. The retinal vasculature and adherentleukocytes were stained by FITC-conjugated concanavalin A (FIG. 12).

Anti-LRP6-1 mAb attenuates ischemia-induced VEGF over-expression in theretina and reduced retinal vascular leakage: Oxygen-induced retinopathy(OIR) is commonly used as a model of DR as it develops both retinalvascular leakage and NV. New born brown Norway rats were exposed to 75%oxygen at the postnatal age of Days 7 to 12 (P7-P12). Anti-LRP6-1 wasinjected into the vitreous at P12, with the same amount of non-specificmouse IgG for control. Retinal vascular permeability was measured usingthe Evans blue albumin leakage method. Evans blue-albumin complex leakedinto the retina was quantified using a spectrophotometer and normalizedby total retinal protein concentration. The results showed that theAnti-LRP6-1 significantly reduced retinal vascular leakage, compared tothe IgG control (FIG. 13A). Since VEGF is known as a major factor for NVand vascular leakage, VEGF levels in the retina were also measured. Asshown by Western blot analysis, retinal VEGF levels were significantlyreduced by Anti-LRP6-1 in OIR rats, compared to those in theIgG-injected OIR rats (FIG. 13B).

Anti-LRP6-1 mAb reduces retinal vascular leakage in diabetic rats:Example 1 showed that the Wnt pathway is also activated in the retina ofSTZ-induced diabetic rats. To evaluate the efficacy of Anti-LRP6-1, saidmAb was injected into the vitreous of STZ-diabetic rats, 2 weeks afterthe onset of diabetes. Retinal vascular leakage was measured 3 weeksafter the mAb injection using Evans blue method. The results showed thatthe rats injected with the mAb had significantly lower retinal vascularleakage compared to those injected with non-specific IgG control (FIG.14), demonstrating that Anti-LRP6-1 reduced retinal vascular leakageinduced by diabetes, and therefore Anti-LRP6-1 has a therapeutic effecton DME.

Anti-LRP6-1 reduces retinal vascular leakage in an AMD model:Vldlr^(−/−) mice are well accepted as a genetic model of wet AMD sincethey develop sub-retinal NV, retina vascular leakage and inflammation.To evaluate the efficacy of Anti-LRP6-1 on AMD, said mAb was injectedinto the vitreous of adult Vldlr^(−/−) mice with the non-specific IgG asa control. Two weeks after the injection, a permeability assaydemonstrated that the eye injected with Anti-LRP6-1 had significantlylower retinal vascular leakage, compared to the IgG control (FIG. 14).This result further demonstrates that Anti-LRP6-1 has a therapeuticeffect on AMD.

EXAMPLE 3

Diabetic retinopathy, a leading cause of blindness, is a severe ocularcomplication involving progressive retinal vascular leakage,neovascularization (NV), and retinal detachment in both type 1 and type2 diabetes. Multiple growth factors, such as vascular endothelial growthfactor (VEGF), play important roles in retinal vascular abnormalities indiabetic retinopathy.

Retinal inflammation in hyperglycemia condition with oxidative stress isbelieved to play a crucial role in the development of diabeticretinopathy. It has been shown that levels of soluble inter-cellularadhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1(VCAM-1), which mediate leukocyte adhesion to the endothelium, weresignificantly increased in the vitreous and serum of diabetic patients.Although numerous observations underscored the association ofhyperglycemia with inflammation in diabetic retinopathy, the pathogenicmechanism leading to chronic inflammation in diabetic retinopathy wasunclear. Recently, the pathogenic role of canonical Wnt signaling inretinal inflammation in diabetic retinopathy has been established.

Wnts, a group of secreted cysteine-rich glycoproteins, initiate asignaling cascade by binding to a receptor complex consisting ofFrizzled (Fz) receptor and low-density lipoprotein receptor-relatedprotein 5/6 (LRP5/6). Upon Wnt ligand binding, C-terminus conservedmotifs (PPPS/TP) of LRP6 are phosphorylated in an orderly fashion by CK1and GSK3β which are recruited to the membrane via scaffold protein Axin.The successive phosphorylation of PPPS/TP motifs reduces cytosolickinase pool, and results in cytosolic β-catenin stabilization andaccumulation. Then, β-catenin is translocated into the nucleus, andassociates with TCF/LEF to activate target gene expression includingVEGF, ICAM-1 and tumor necrosis factor-α (TNF-α).

Example 1 showed that retinal levels of β-catenin are increased inhumans with diabetic retinopathy and in diabetic retinopathy animalmodels. Retinal levels of LRP6 were also increased in the retina ofdiabetic retinopathy models. In addition, an intravitreal injection of aWnt antagonist Dickkopf homolog 1 (DKK1) effectively ameliorateddiabetic retinopathy in animal models, indicating a key role of thedysregulation of the Wnt pathway in the pathogenesis of diabeticretinopathy. Furthermore, activation of Wnt signaling alone in theretina of normal rats is sufficient to induce pathological changes suchas retinal inflammation and vascular leakage. Recent evidence alsoindicated that Wnt signaling is directly linked to the inflammatoryresponses in general, as it was documented that Wnt signaling activatesnuclear factor κB (NF-κB) and up-regulates expression of a number ofinflammatory factors including signal transducer and activator oftranscription 3 (STAT-3). Together, these previous findings indicatethat over-activation of the Wnt pathway plays a pathogenic role indiabetic retinopathy and represents a promising drug target for thetreatment of diabetic retinopathy.

The Wnt co-receptor LRP6 plays a pivotal role in the Wnt signalingactivation. It provides binding sites for both Wnt ligands andantagonist DKK1 on its ectodomain. Moreover, its conserved intracellularPPPS/TP motifs are essential docking sites for protein complexAxin/GSK3β for signal amplification, which is sufficient to transmitsignals from Wnt ligands to the intracellular cascade. These factsindicate that LRP6 represents an ideal target for blocking the Wntpathway.

Numerous pharmaceutical antibodies have been reported to not only targetspecific oncogenic receptors such as epidermal growth factor receptor,but also to neutralize soluble proteins such as VEGF in multiple diseasemodels. To assess the impact of blockade of LRP6 on the Wnt signalingpathway activity and retinal inflammation in diabetic retinopathy, amonoclonal antibody specific for the extracellular domain of LRP6including the first and second propeller domains was generated. Itsinhibitory effects on the canonical Wnt pathway and its beneficialeffects on diabetic retinopathy were further evaluated using diabeticmodels.

Materials and Methods of Example 3

Plasmids, antibodies, and reagents: The plasmids expressing thefull-length LRP6, ectodomain of LRP6 (LRP6N), ectodomain of LRP5(LRP5N), and ectodomain of low-density lipoprotein receptor (LDLRN) werekind gifts from Dr. X. He at Harvard University. The human cDNA ofdeletion mutants of LRP6, LRP6E1E2 and LRP6E3E4, were amplified bypolymerase chain reaction and tranferred into pET28b+ plasmid forexpression in BL21(DE3). Human full-length LRP5 and VLDLR-N weresubcloned into pGEMT-easy vector and then pcDNA3.1(−) and pcDNA6plasmids, respectively, for expression in mammalian cells. The humanWnt1 cDNA was PCR-amplified and cloned into pcDNA6 plasmid forexpression.

Mouse anti-β-actin antibody, rabbit anti-TNF-α antibody (Abcam,Cambridge, Mass.), mouse anti-VEGF antibody, goat anti-ICAM-1 antibody,rabbit anti-β-catenin antibody (Santa Cruz Biotechnologies, Santa Cruz,Calif.), rabbit anti-pLRP6 (at Ser1490) antibody, rabbitanti-p-β-catenin (Ser33/37/Thr41) antibody (Cell Signaling, Danvers,Mass.) were used for Western blot analysis. Monoclonal antibodyAnti-LRP6-1 was purified using protein G column (Thermo FisherScientific Inc., Waltham, Mass.) following manufacturer's instruction.

D-glucose, lithium chloride, sodium chloride were purchased fromSigma-Aldrich Inc. (St. Louis, Mo.). Non-specific mouse IgG waspurchased from Vector laboratories (Burlingame, Calif.).

Cell culture, transfection, and antibody treatment: hTERT-RPE (ATCC,Manassas, Va.), rat Miller (rMC-1; a kind gift from Dr. Vijay Sarthy atNorthwestern University), and bovine retinal capillary endothelial cells(RCECs) were maintained in Dulbecco's modified Eagle's medium (DMEM;Cellgro, Manassas, Va.) supplemented with 10% FBS (Invitrogen, Carlsbad,Calif.). Mouse L cells stably expressing Wnt3A were maintained in DMEMsupplemented with 10% calf FBS, and Wnt3A conditioned media (WCM) wereused as a source of Wnt ligand. Lipofectamine 2000 (Invitrogen,Carlsbad, Calif.) was used for the transfection of TOP FALSH promoterconstructs and expression plasmids following the protocol recommended bythe manufacturer. Cells were synchronized in serum-free media for 3hours, and were treated with different concentrations of Anti-LRP6-1 for0.5 hour, supplemented with non-specific mouse IgG to the equalconcentrations of total IgG in each well. Then, the cells were treatedwith 20% WCM or 30 mM D-glucose to activate Wnt signaling.

Conditioned media containing VLDLRN with a 6× histidine tag(VLDLRN-his), LDLRN with a myc tag (LDLRN-myc), LRP5N-myc and LRP6-mycwere obtained following the procedures. HEK-293T cells at 60% confluencywere transiently transfected with expression plasmids usinglipofectamine 2000 (Invitrogen, Carlsbad, Calif.), and culture mediawere collected after 48 hours incubation. The media were filtered andused as a conditioned media (CM).

Antigen preparation, monoclonal antibody selection, and purification:LRP6E1E2 and LRP6E3E4 were subcloned into pGEMT-easy vector bypolymerase chain reaction from human origin cDNA, and transferred topET28b+ plasmid. Then, pET28b+ containing LRP6E1E2 and LRP6E3E4sequences were transformed into BL21(DE3) for expression. The inclusionbody was purified and then dissolved in a buffer containing 6 Mguanidine hydrochloride. The denatured proteins were purified usingNi-NTA resin (Novagen, Madison, Wis.) following manufacturer'sinstruction. Purified antigens were confirmed by Coomassie blue stainingand Western blot analysis with an anti-His tag antibody.

On-column refolding was performed by serial buffer changes. Ten columnvolumes (CV) of buffer A (0.1% Triton X-100, GSH/GSSG in PBS, pH 7.8),10 CV of buffer B (5 mM beta-cyclodextrin, GSH/GSSG in PBS, pH 7.8), andwashing buffer (20 mM imidazole in PBS, pH 7.8) were serially used forrefolding. The protein was eluted with 300 mM imidazole in phosphatebuffered saline (PBS). The eluates were dialyzed in PBS, and proteinconcentration was measured by Bradford assay.

Monoclonal antibodies for LRP6 ectodomain were generated via acontracted service by Proteintech Group (Chicago, Ill.) using thepurified antigen. Among various positive clones screened out from ELISA,Anti-LRP6-1 was identified to be specific and to have blocking activity,by specificity tests using ELISA and Western blot analysis.

Anti-LRP6-1 used in cell culture and intra-vitreal injection wasproduced and purified following the conventional methods (Hendriksen etal., Res immunol, 149:535-542 (1998)). Briefly, 6 wks-old female BALB/Cmouse abdominal cavity was injected with hybridoma cells (1×106/ml) andascites were collected after 7 days post-injection. The collected mouseascites were applied onto a protein G column following manufacturer'sprotocol. The mAb eluates from the column were dialyzed against PBSovernight.

Luciferase reporter assays: hTERT-RPE cells, plated at 2×10⁴ cells/wellin 24-well plates, were transfected with 0.25 μg TOPFlash and 0.05 μgpRL-TK constructs using lipofectamine 2000. Four hourspost-transfection, the culture media was replaced by fresh media and thecells were pre-treated with Anti-LRP6-1, with supplemented amount ofcontrol IgG to reach the same concentration of total IgG in each well,for 0.5 hours and then Wnt3A CM was added for 16 hours. For the analysisusing Wnt1, expression plasmid of Wnt1 was transfected with promoterconstructs, and followed by the same procedures. Luciferase activitymeasurement was performed using dual luciferase assay kit (Promega,Madison, Wis.) following the manufacturers' protocols. Renillaluciferase activity was measured to normalize transfection efficiency.All experiments were performed at least in triplicate.

Western blotting: Cultured cells were washed with cold phosphatebuffered saline (PBS) and lysed in cell lysis buffer (150 mM NaCl, 1%NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0, 10 mM sodiumpyrophosphate, 100 mM sodium fluoride, and 2 mM phenylmethylsulfonylfluoride). After washing with PBS, the cells were suspended in PBS witha protease inhibitor cocktail (Roche Applied Science, Indianapolis,Ind.) and two times of freeze/thaw cycles were performed to isolate thecytosolic fraction. The same amounts of retinal proteins from eachanimal were obtained following sonication in the lysis buffer (same asabove) and used for Western blot analysis after total proteinconcentration measurement by Bradford assay. Further, specific primaryantibodies for each protein were incubated overnight and HRP-conjugatedsecondary antibody was used for detection. The signal was developed witha chemiluminescence detection kit (ECL; Amersham International,Piscataway, N.J.). Blots were then stripped and re-blotted with anantibody specific for β-actin for loading control. Images were capturedby a Chemi Genius Image Station (SynGene, Frederick, Md.). Individualprotein band was semiquantified by densitometry using the GENETOOLSprogram (SynGene, Frederick, Md.).

Immunohistochemistry: Immunostaining was performed as describedpreviously (Zhang et al., Am J Pathol, 166:313-321 (2005)). Briefly, theeyes were cross-sectioned sagittally through the center of the corneaand optic nerve head, and both halves of the eyeball were embedded withthe center facing down. Serial cryosections (5 μm thickness) wereblocked with 1% BSA in PBS and incubated with FITC-conjugatedAnti-LRP6-1, with FITC-IgG as a control. For negative control,FITC-Anti-LRP6-1 was pre-adsorbed with antigen, LRP6E1E2 peptide, todemonstrate the specificity the antibody staining. After PBS-0.1%tween-20 washes, the slides were then rinsed in PBS and viewed under afluorescence microscope.

Rat models of OIR and STZ-induced diabetes. All of the animalexperiments were performed in compliance with the ARVO Statement for theUse of Animals in Ophthalmic and Vision Research. Brown Norway rats(Charles River Laboratories, Wilmington, Mass.) were used for the OIRmodel following an established protocol (Ricci, Doc Opthalmol,74:171-177 (1990)). Diabetes was induced and monitored in adult BrownNorway rats by an intraperitoneal injection of STZ (55 mg/kg in 10mmol/L of citrate buffer, pH 4.5) as described previously (Zhang et al.,Am J Pathol, 166:313-321 (2005)). Diabetic rats at 2 weeks after theonset of diabetes received an intravitreal injection of Control IgG orAnti-LRP6-1.

Intravitreal injection: Animals were anesthetized with a 50:50 mix ofketamine (100 mg/mL) and xylazine (20 mg/mL), and pupils were dilatedwith topical application of phenylephrine (2.5%) and tropicamide (1%). Asclerotomy was created approximately 1 mm posterior to the limbus with aneedle (˜32 gauge). A glass injector (33 gauge) connected to a syringefilled with 2 μL of LRP6 mAb with desired concentrations or purifiedmouse IgG was introduced through the sclerotomy into the vitreouscavity.

Retinal vascular permeability assay: Retinal vascular permeability wasmeasured using Evans blue-albumin as tracer following an establishedprotocol (Aiello et al., Am J Opthalmol, 132:760-776 (2001)).Concentrations of Evans blue in the retina were normalized by totalretinal protein concentrations and by Evans blue concentrations in theplasma.

Leukostasis assay: The assay was performed as described previously (Chenet al., Microvasc Res, 78:119-127 (2009)). Briefly, mice wereanesthetized and perfused through the left ventricle with PBS to removecirculating leukocytes in blood vessels. The adherent leukocytes in thevasculature were stained by perfusion with FITC-conjugatedconcanavalin-A (Con-A, 40 μg/ml, Vector Laboratories, Burlingame,Calif.). The eyes were removed and fixed in 4% paraformaldehyde. Theretinas were dissected and flat-mounted. Adherent leukocytes in theretinal vasculature were counted under a fluorescence microscope.

Statistical analysis: The quantitative data were analyzed and comparedusing Student's t test. Statistical significance was set at p<0.05.

Results of Example 3

Specificity of Anti-LRP6-1 for LRP6 E1E2 domain: A number of clones ofmAb were raised using the recombinant ectodomain of LRP6. Out ofnumerous positive clones of mAb, Anti-LRP6-1 was selected based on itsspecificity for endogenous LRP6 in various cell lines. Anti-LRP6-1recognized LRP6 as a single band, but not LRP5, the other Wntco-receptor with 71% amino acid sequence identity to LRP6 in the human.Furthermore, Anti-LRP6-1 recognized endogenous LRP6 in various celllines from different species including the human, mouse, rat and bovine(FIG. 15A-B). As LRP6 belongs to the low-density lipoprotein receptor(LDLR) family, the possible binding of Anti-LRP6-1 to other members ofthe LDLR family such as LDLR and very low-density lipoprotein receptor(VLDLR) was evaluated (FIG. 15C). Western blot analysis showed thatAnti-LRP6-1 did not recognize LDLR or VLDLR, indicating its specificityfor LRP6. Furthermore, it was confirmed that Anti-LRP6-1 recognized thefull-length ectodomain and the E1E2 fragment of LRP6, but not the E3E4domain, indicating that the epitope of Anti-LRP6-1 is present in theE1E2 domain of LRP6 (FIG. 15C).

Next, rat and mouse retinal sections were immunostained withFITC-labeled Anti-LRP6-1 to determine spatial expression of LRP6 in theretina. The immunohistochemistry using FITC-Anti-LRP6-1 showed that LRP6is primarily expressed in the inner retina in rats. In the mouse retina,however, intensive LRP6 signal was observed in the inner retina as wellas in the RPE (FIG. 15C). Immunostaining with the isotype control(non-specific IgG) (D1, D4, D7, D10) and FITC-Anti-LRP6-1 pre-absorbedby antigen (LRP6 ectodomain peptide) (D3, D6, D9, D12) under the sameconditions did not show specific signals, indicating that the LRP6signals are specific.

Anti-LRP6-1 efficiently inhibited Wnt ligand-induced Wnt signaling atthe receptor level. To reveal if Anti-LRP6-1 has an inhibitory effect onthe canonical Wnt pathway, hTERT-RPE, a cell line derived from humanretinal pigment epithelial (RPE) cells and expressing endogenous LRP6,was exposed to Wnt3A conditioned media or was transfected with Wnt1 toactivate the canonical Wnt pathway. Anti-LRP6-1 efficiently inhibitedWnt3A-mediated phosphorylation of LRP6 on residue Serine1490, an earlystep in Wnt pathway activation, while having no effect on total LRP6levels. Anti-LRP6-1 also attenuated the Wnt ligand-induced increase ofcytosolic β-catenin levels, indicating attenuation of β-cateninaccumulation. In contrast, negative control IgG had no effect on pLRP6and cytosolic-β-catenin levels (FIG. 16A). The effect of Anti-LRP6-1 ontranscriptional activity of TCF/β-catenin was evaluated using TOPFLASHactivity assay which measures Luciferase activity driven by a promotercontaining TCF/β-catenin-binding sites. Wnt3A induced Luciferaseactivity by 40 folds, and the Anti-LRP6-1 suppressed the Wnt3A-inducedLuciferase activity in a dose-dependent manner with EC50 approximately20 μg/ml. Similarly, Anti-LRP6-1 also inhibited the Wnt1-inducedTOPFLASH activity with EC50 approximately 20 μg/ml (FIG. 16C).

Lithium chloride (LiCl) is a known inhibitor of GSK3β thatconstitutively phosphorylates β-catenin, which mediates proteasomaldegradation of β-catenin. Thus, lithium activates canonical Wntsignaling independent of Wnt ligands, Wnt receptors and co-receptors. Asshown by TOPFLASH assay, 25 mmol/L LiCl induced TCF/β-catenin activitymarkedly. Anti-LRP6-1 did not inhibit lithium-induced TOPFLASH activity,indicating the inhibition by Anti-LRP6-1 is at the level ofreceptor-ligand interactions (FIG. 16D).

Anti-LRP6-1 inhibited high-glucose (25 mM)-activated canonical Wntsignaling. It has been documented that the canonical Wnt pathway isactivated in the retinae of diabetic patients and animal models, andhigh glucose medium also activates Wnt signaling in the cultured cells.Retinal cells relevant to the pathogenesis of diabetic retinopathy,including RPE, Müller and endothelial cells, were exposed to a highglucose medium (30 mM) to activate the Wnt pathway, with low glucosemedium (5 mM glucose and 25 mM mannitol) as control. Anti-LRP6-1 wasadded to the medium to determine its inhibitory effect on highglucose-induced Wnt signaling. Western blot analysis showed that highglucose exposure for 6 hours increased p-LRP6 and β-catenin levels inthe RPE cells. Anti-LRP6-1 sufficiently attenuated the highglucose-induced increases of p-LRP6 and β-catenin accumulation in aconcentration-dependent manner (FIG. 17A-B). To further confirm theactivation of Wnt signaling, expression levels of Wnt target genes suchas cyclin D1 and c-myc were measured. High glucose medium up-regulatedexpression of cyclin D1 and c-myc as shown by Western blot analysis;however, Anti-LRP6-1 attenuated the levels of cyclin D1 and c-myc (FIG.17C).

Retinal Miller cells are known to play a key role in retinalinflammation in diabetic retinopathy. In Müller cells, Anti-LRP6-1 alsoattenuated the high glucose-induced increase of cytosolic β-cateninlevels (FIG. 17D). As retinal endothelial cells are major players inretinal vascular leakage, leukostasis and neovascularization in diabeticretinopathy, bovine retinal capillary endothelial cells (BRCEC) wereexposed to the 30 mM D-glucose to activate Wnt signaling. Compared tolow glucose control (5 mmol/L glucose and 25 mmol/L mannitol), 30 mMD-glucose increased cytosolic β-catenin, while Anti-LRP6-1 inhibited theincrease induced by high glucose in BRCEC. Since phosphorylation ofβ-catenin by GSK3β (Ser33/37/Thr41) leads to degradation of β-catenin,measured phosphorylated β-catenin levels were also measured. The resultsshowed high glucose significantly decreased p-β-catenin levels, whileAnti-LRP6-1 reversed the change induced by high glucose (FIG. 17E).

In summary, these results showed that high glucose activated the Wntsignaling pathway and its target gene expression in retinal cellsincluding RPE, Müller cells, and BRCEC, while Anti-LRP6-1 attenuated thehigh glucose-induced Wnt signaling in a concentration-dependent fashion.

Anti-LRP6-1 suppressed the high glucose-induced expression ofinflammatory and angiogenic factors. Over-expression of angiogenic andinflammatory factors in the retina is known to play pathogenic roles inretinal neovascularization and inflammation in diabetic retinopathy.Many of these inflammatory factors are regulated by Wnt signaling. Ithas been previously described that VEGF is a direct target regulated bythe canonical Wnt signaling. In cultured RPE, Miller cells and BRCEC,high glucose medium induced over-expression of VEGF, while Anti-LRP6-1blocked the over-expression in a concentration-dependent manner.Similarly, high glucose also induced over-expression of inflammatoryfactors ICAM-1 and TNF-α. Anti-LRP6-1 suppressed the over-expression ofthese factors in the retinal cells (FIG. 18). In the same cells,non-specific IgG did not reduce the levels of these factors. Anti-LRP6-1at high concentrations reduced the levels of VEGF, ICAM-1 and TNF-α to arange of low glucose control (FIG. 18). These results indicate that highglucose induces over-expression of these factors through Wnt signaling.

Anti-LRP6-1 inhibited endothelial cell migration. The effect ofAnti-LRP6-1 on endothelial migration was evaluated by scratch woundhealing assay and tube formation assay using primary BRCEC, asendothelial cell migration is an important step in retinalneovascularization. The scratch wound healing assay showed that highglucose medium enhanced BRCEC wound healing 48 hours after the scratch.In the presence of Anti-LRP6-1, the high glucose induced BRCEC woundhealing was substantially decreased (FIG. 19A-B). In BRCEC tubeformation assay, BRCEC formed a tube like pattern in 12 hours, which wasattenuated by Anti-LRP6-1, but not by IgG (FIG. 19C-D). Taken together,these results demonstrated that Anti-LRP6-1 inhibited endothelial cellmigration.

Anti-LRP6-1 reduced vascular leakage and inhibited inflammation in theretina of OIR model. One of the hallmarks of diabetic retinopathy is aleaky vessel due to breakdown of the blood-retina barrier. OIR rats, amodel of proliferative diabetic retinopathy, manifest increased retinalvascular permeability due to ischemia-mediated over-expression ofpro-angiogenic factors such as VEGF, a target gene of Wnt signaling.Inhibitory effect of Anti-LRP6-1 on vascular leakage was evaluated inthe OIR model. Anti-LRP6-1 was injected intravitreally into the righteye (10 μg/eye) at age P12 and the same amount of control IgG into thecontralateral eyes. Retinal vascular leakage was measured using theEvans blue-albumin leakage method at P16, which showed that the eyesinjected with Anti-LRP6-1 had significantly lower retinal vascularpermeability compared to that injected with control IgG (FIG. 20A).Next, retinal levels of the factors contributing to vascular leakage andinflammation were measured. Compared to control IgG, Anti-LRP6-1suppressed the expression of ICAM-1, TNF-α, and VEGF in the retina ofOIR rats. Anti-LRP6-1 also down-regulated retinal levels of LRP6 andβ-catenin, indicating that Anti-LRP6-1 attenuatedangiogenic/inflammatory activities in the retina of OIR via inhibitingWnt signaling activation (FIG. 20B-D).

Anti-LRP6-1 reduced retinal vascular leakage and leukostasis inSTZ-induced diabetic rats. To evaluate the beneficial effect ofAnti-LRP6-1 on diabetic retinopathy, Anti-LRP6-1 was intravitreallyinjected into STZ-induced diabetic rats, with the same dose ofnon-specific IgG for control. Retinal vascular permeability was measuredtwo weeks following the Anti-LRP6-1 injection using the Evansblue-albumin leakage method, and compared to the IgG control. The resultdemonstrated that the eyes injected with Anti-LRP6-1 had significantlylower vascular permeability than that injected with IgG (FIG. 21A).

Retinal inflammation such as leukostasis is another hallmark of diabeticretinopathy. To determine the effect of Anti-LRP6-1 on retinalinflammation, a leukostasis assay was performed two weeks following themAb injection in STZ-diabetic retina. Compared to non-diabetic control,untreated STZ-diabetic and IgG-treated STZ-diabetic retinae showedsignificantly increased numbers of adherent leukocytes in retinalvasculature (FIG. 21B). The number of leukocytes was significantlydecreased in the STZ-diabetic group injected with Anti-LRP6-1,indicating that Anti-LRP6-1 has inhibitory effect on retinalinflammation (FIG. 21B).

As ICAM-1 and TNF-α play important roles in retinal inflammation underdiabetes, retinal levels of ICAM-1 and TNF-α were further measured usingWestern blot analysis. The STZ-induced diabetic retina showedover-expression of ICAM-1 and TNF-α, compared to that in non-diabeticrats. Anti-LRP6-1 but not the non-specific IgG, suppressed theover-expression of ICAM-1 and TNF-α in the diabetic retina (FIG. 21C).In addition, retinal levels of LRP6 were increased in the STZ-induceddiabetic retina, compared to that in non-diabetic rats. However, thelevels were decreased in the diabetic rats injected with Anti-LRP6-1,but not in that injected with non-specific IgG. These data indicate thatactivation of the Wnt pathway in the diabetic retina was in part due toover-expression of LRP6, and Anti-LRP6-1 suppresses over-expression ofICAM-1 and TNF-α through down-regulation of LRP6. Taken together, theresults demonstrated that Anti-LRP6-1 has beneficial effects on diabeticretinopathy as it attenuates vascular leakage and inflammation in thediabetic retina.

Discussion of Example 3

The previous Examples show dysregulation of Wnt signaling in the retinawith diabetic retinopathy and thus indicate that Wnt signaling is amajor pathogenic pathway in diabetic retinopathy. The pathogenic role ofWnt signaling in diabetic retinopathy is supported by the observationthat DKK1, a specific inhibitor of Wnt signaling, can ameliorate retinalinflammation, vascular leakage and inflammation in diabetic retinopathymodels. These findings indicate that Wnt signaling represents apotential therapeutic target of diabetic retinopathy. Despite thewell-studied molecular cascade of Wnt signaling, an effective strategyto block the Wnt pathway has not been established for therapeuticapplication of diabetic retinopathy. Although natural inhibitors of theWnt signaling such as DKK family members, SERPINA3K and IGF1BP have beenidentified, there is limitation using these natural inhibitors,including low stability and high costs of production, in theirapplications as therapeutic compounds. The present Example reports forthe first time that an anti-LRP6 monoclonal antibody attenuates retinalvascular leakage and retinal inflammation in diabetic retinopathy viainhibition of the Wnt/β-catenin signaling. In addition, these resultsprovide further support for a causative role of Wnt pathway activationin the development of diabetic retinopathy and indispensability of LRP6in this context. These observations firstly established that blockingLRP6 by the Anti-LRP6-1 can ameliorate diabetic retinopathy, indicatingits therapeutic potential. Moreover, this Example reveals that LRP6 as asufficient target for blocking the Wnt signaling pathway.

Here, it is demonstrated that Anti-LRP6-1 specific for the first andsecond propeller domains of LRP6 (E1E2) inhibited Wnt signaling as wellas subsequent expression of angiogenic and inflammatory factors invarious retinal cells. Based on its inhibitory effect on Wnt signalingand inflammatory factors in vitro, the potential beneficial effects ondiabetic retinopathy was determined in animal models. First, in both OIRand diabetic rats, the mAb reduced retinal vascular leakage that is theprimary cause of diabetic macular edema (DME), the number one cause ofvision loss in diabetic patients. Second, the Anti-LRP6-1 suppressedretinal leukostasis, a key inflammatory change that can lead to impairedendothelium, vascular leakage and closure of capillary whichsubsequently results in local ischemia. Toward the mechanism for itseffects on retinal vascular leakage and leukostasis, these in vitro andin vivo results both showed that Anti-LRP6-1 down-regulates expressionof VEGF, ICAM-1 and TNF-α which have been shown to play key roles inretinal inflammation in diabetes. VEGF has been well established as atarget gene of Wnt signaling. Sequence analysis of the promoter regionsof ICAM-1 and TNF-α revealed that there are TCF/LEF binding sites in adistal region of their promoters, indicating that expression of ICAM-1and TNF-α may be directly regulated by the Wnt pathway.

The canonical Wnt pathway is a conserved signaling pathway that utilizessingle effector, multi-functional transcription activator β-catenin, toregulate expression of a number of target genes. However, diversespatiotemporal activation of the Wnt pathway arises from multiplecombinations among 20 Wnt ligands, 10 Frizzled receptors, and 2coreceptors, providing numerous diversities. These diversities dampenthe therapeutic approaches to inhibit the Wnt pathway via blockage ofWnt ligands or Frizzled receptors. Based on the following facts,however, LRP6 is believed to be an ideal target for blocking Wntsignaling. 1) The canonical Wnt pathway requires one of the twoco-receptors, LRP5 or LRP6. 2) Knockout of LRP6 manifests more severephenotypes than knockout of LRP5, indicating LRP6 plays a more importantrole than LRP5 in Wnt signaling. 3) LRP6 has a large extracellulardomain which is accessible extracellularly by antibodies. In addition,the studies outlined in Example 1 from diabetic patients and animalmodels clearly demonstrated that blocking LRP6 by DKK1 can amelioratediabetic retinopathy in animal models. Although the exact mechanism forWnt signaling activation in diabetes is obscure, the present andprevious studies indicated that phosphorylation of LRP6 by lipidoxidation product (4-hydroxynonenal) and by high glucose were sufficientfor Wnt signaling activation. Wnt ligand-mediated signaling seems to bethe secondary effect in this specific condition. These findings stronglyindicate that blocking LRP6, rather than Wnt ligands or Frizzledreceptors, provides more effective means to inhibit at least thecanonical pathway in diabetic condition.

LRP6 is a type 1 single transmembrane receptor, whose larger ectodomainis composed of four similar EGF-like repeats (E1-E4) with YWTD propellerdomain. It has been indicated that LRP6E1E2 domain including the firstand second beta-propeller regions cooperates to interact with Wnt-Fz,whereas LRP6E3E4 provides binding site for antagonist DKK1. However,recent in vitro reconstitution studies have shown that Wnt3A binds tothe E3E4 domain specifically, and DKK1 to both E1E2 and E3E4 with acooperative pattern of interactions. Also, it is further determined thatWnt1 preferentially binds to E1 domain, and E3 is sufficient for Wnt3Abinding. Despite their different binding sites on LRP6, both Wnt1 andWnt3A-induced TOPFLASH activities are inhibited by Anti-LRP6-1,indicating that Anti-LRP6-1 not only blocks Wnt1 ligand binding to theE1 domain, but also has inhibitory effect on Wnt3A interaction to E3domain. The inhibition of Anti-LRP6-1 on Wnt3A-induced Wnt signaling maybe explained by a possible mechanism that the bivalent Anti-LRP6-1binding to LRP6 changes the three dimensional structure of LRP6ectodomain which causes inaccessibility of Wnt3A. It is also likely thatAnti-LRP6-1 may destabilize LRP6 which causes down-regulation of LRP6available for Wnt ligand binding.

The in vivo studies using OIR and STZ-induced diabetic rat models showedthat Anti-LRP6-1 not only inhibits the activation of the canonical Wntpathway, but also down-regulates LRP6 levels, as retinal levels of totalLRP6 are lower in the rats injected with the Anti-LRP6-1 (1 week afterthe injection) than that injected with the control IgG. However, thecell culture results showed that Anti-LRP6-1 blocks the activation ofLRP6 but does not decrease total LRP6 levels after 6 hours treatment.This disparity between in vitro and in vivo results may be explained bythe different treatment times by Anti-LRP6-1, since the cultured cellswere treated with Anti-LRP6-1 for 6 hours while the retina was treatedfor 1 week in diabetic rats. The mechanism by which Anti-LRP6-1down-regulates total level of LRP6 in OIR and diabetic animal models isunclear. However, it is likely that antibody binding to the ectodomainof LRP6 may destabilize LRP6 or induce its internalization and thus,decrease total level of LRP6.

The apparent involvement of Wnt signaling in the regulation of multiplepathogenic mechanisms involved in diabetic retinopathy makes the Wntpathway an intriguing candidate for targeted therapy. VEGF, whichregulates angiogenesis, vascular permeability, and migration ofendothelial cells, is over-expressed in the retina of diabeticretinopathy and is a Wnt target gene regulated by β-catenin. ThisExample showed that Anti-LRP6-1 suppressed over-expression of VEGF inretinal cells exposed to high-glucose and in the retina of OIR rats. Inaddition, the Wnt pathway is known to regulate retinal inflammationthrough direct activation of NF-κB or crosstalk with transcriptionfactors including STAT3. The results also indicated that temporalexpression of ICAM-1 and TNF-α was in parallel with VEGF expression uponWnt signaling activation, providing supplementary evidence that theirexpression is under the control of Wnt signaling. Thus, these findingsprovide possible evidence that expression of multiple factors known toplay important pathogenic roles in diabetic retinopathy aredown-regulated by the Wnt pathway.

Recent clinical studies showed that anti-VEGF compounds have promisingeffect on AMD. In diabetic retinopathy, however, the anti-VEGF compoundsare not as effective as in AMD. A possible reason is that diabeticretinopathy is a complex, and multifactorial disorder. Multiple growthfactors, in addition to VEGF, are known to play roles in diabeticretinopathy. Therefore, blocking VEGF alone may not be sufficient forameliorating diabetic retinopathy. Since the Wnt pathway regulatesmultiple inflammatory and angiogenic factors, such as ICAM-1, PDGF,VEGF, TNF-α, MMP and COX2 which are implicated in diabetic retinopathy,the anti-LRP6 antibody may have a more potent efficacy than anti-VEGFcompounds in diabetic retinopathy.

Advantages of using humanized monoclonal antibodies for therapeuticpurpose are their high specificity and stability, and low immunogenecityand toxicity, compared to endogenous Wnt inhibitors. It is noteworthythat the present study is simply a proof of concept study. The antibodyneeds to be humanized. The efficacy, toxicity and immunogenicity willneed to be determined.

In conclusion, these findings firstly demonstrated that anti-LRP6monoclonal antibody has therapeutic potential in diabetic retinopathy.Also, the data implicate LRP6 activation as a major cause of retinalvascular leakage and retinal inflammation, thus expanding involvement ofLRP6 and targeted therapy in AMD which proceeded by similar pathogenicmechanisms. Furthermore, these results indicate that the Anti-LRP6-1decreases the total level of LRP6, indicating its antagonistic effectregardless of different Wnt ligands. Even though questions for sideeffect on cell survival, stability issue, and efficacy remain, itfirstly demonstrated the therapeutic potential of LRP6 targeting therapyin diabetic retinopathy. Further, this study provides a strong rationalefor investigating antibody-based, LRP6-targeted therapies in diseasesassociated with Wnt signaling activation and/or over-expression of LRP6.

EXAMPLE 4

Effect of Anti-LRP6-1 on CTGF (factor involved in fibrosis): Theanti-LRP6 mAb attenuates the hypoxia-induced over-expression of ICAM-1and CTGF. ICAM-1 is a major adhesion molecular on the endothelium andresponsible for leukostasis and endothelium damage in diabetes. CTGF isa fibrogenic factor and is responsible for the basement membranethickening in DR. Thus, the effect of Anti-LRP6-1 on the expression ofICAM-1 and CTGF was measured. Hypoxia was induced in ARPE19 cells usingCoCl₂, which induced significant over-expression of ICAM-1 and CTGF, asshown by Western blot analysis (FIG. 22). Anti-LRP6-1 attenuated theover-expression of ICAM-1 and CTGF in a concentration-dependent manner(FIG. 22).

Effect of Anti-LRP6-1 on choroidal neovascularization (wet-AMD): The Wntpathway is activated in the eyecups with laser-induced CNV, which isattenuated by Anti-LRP6-1. CNV was induced by laser photocoagulation inBN rats. A group of CNV rats received an intravitreal injection ofAnti-LRP6-1 and another group received the same amount of non-specificrat IgG, at the same day as the laser. At day 7 after the laser, therats were perfused and eyecups (retina, RPE and choroid) were isolatedfor Western blot analysis using an antibody for β-catenin. The resultsshowed that β-catenin was up-regulated in the eyes with laser-CNV,compared to the eyes without laser. Injection of Anti-LRP6-1significantly decreased β-catenin levels, compared to IgG control (FIG.23). As accumulation of β-catenin is a key step in activation of Wntsignaling, these results indicate that laser-induced CNV activates theWnt pathway, while Anti-LRP6-1 attenuates Wnt signaling activation inthe CNV eyes.

Anti-LRP6-1 ameliorates laser-induced CNV. The effect of Anti-LRP6-1injection on laser induced CNV was examined by fluorescein angiography,and the severity of CNV was evaluated by quantifying Grade 4 lesions infundus images. The result showed that Anti-LRP6-1 significantlydecreased numbers of Grade 4 lesions, compared to the control eyesinjected with non-specific murine IgG (FIG. 24), indicating a beneficialeffect on subretinal vascular leakage and CNV.

Anti-LRP6-1 decreases area of laser-induced CNV. Anti-LRP6-1 wasinjected intravitreally into the eyes with laser-induced CNV at the sameday of the laser, with the same amount IgG as control. Two weeks afterthe injection, CNV lesions were visualized by fluorescein angiography onthe flat-mounted retina-choroid complex. The areas of CNV were measuredby computer-assisted image analysis and averaged. The results showedthat the Anti-LRP6-1 injected group had significantly decreased CNVareas, compared to that injected with IgG and that without injection(FIG. 25). These results indicate that Anti-LRP6-1 inhibitslaser-induced CNV in the rat model.

Thus, in accordance with the presently disclosed inventive concept(s),there has been provided monoclonal antibodies for blocking the Wntsignaling pathway, as well as methods for producing and using same.Although the presently claimed and disclosed inventive concept(s) hasbeen described in conjunction with the specific drawings and languageset forth above, it is evident that many alternatives, modifications andvariations will be apparent to those skilled in the art. Accordingly, itis intended to embrace all such alternatives, modifications andvariations that fall within the spirit and broad scope of the inventiveconcept(s).

What is claimed is:
 1. A method of inhibiting and/or decreasing theoccurrence and/or severity of at least one condition selected from thegroup consisting of inflammation, vascular leakage, fibrosis, abnormalneovascularization, and cancer, said method comprising the step of:administering to a subject suffering from or predisposed to the at leastone condition a pharmaceutical composition, wherein the pharmaceuticalcomposition inhibits activation of the Wnt signaling pathway, therebyinhibiting and/or decreasing the occurrence and/or severity of the atleast one condition, and wherein the pharmaceutical compositioncomprises a monoclonal antibody or antigen binding fragment thereof anda pharmaceutically acceptable carrier, and wherein the monoclonalantibody or antigen binding fragment thereof comprises: a heavy chainvariable region CDR1 having the amino acid sequence of SEQ ID NO: 9, aheavy chain variable region CDR2 having the amino acid sequence of SEQID NO: 11, a heavy chain variable region CDR3 having the amino acidsequence of SEQ ID NO: 13, a light chain variable region CDR1 having theamino acid sequence of SEQ ID NO: 15, a light chain variable region CDR2having the amino acid sequence of SEQ ID NO: 17, and a light chainvariable region CDR3 having the amino acid sequence of SEQ ID NO: 19;and wherein the monoclonal antibody or antigen binding fragment thereofspecifically binds to an epitope within the LRP6 extracellular domain,wherein the LRP6 extracellular domain has the amino acid sequence of SEQID NO:
 2. 2. The method of claim 1, wherein the monoclonal antibody orantigen binding fragment thereof has: a heavy chain variable regionamino acid sequence that is at least 90% identical to SEQ ID NO: 5, andwherein the CDR1, CDR2, and CDR3 thereof are 100% identical to the aminoacid sequences of SEQ ID NOS:9, 11, and 13, respectively; and a lightchain variable region amino acid sequence that is at least 90% identicalto SEQ ID NO: 7, and wherein the CDR1, CDR2, and CDR3 thereof are 100%identical to the amino acid sequences of SEQ ID NOS:15, 17, and 19,respectively.
 3. The method of claim 1, wherein the monoclonal antibodyor antigen binding fragment thereof is selected from the groupconsisting of a full length immunoglobulin molecule, an scFv, a Fabfragment, an Fab′ fragment, an F(ab′)2, an Fv, a disulfide linked Fv,and combinations thereof.
 4. The method of claim 1, wherein themonoclonal antibody or antigen binding fragment thereof is humanized. 5.The method of claim 1, wherein the monoclonal antibody or antigenbinding fragment thereof binds to the LRP6 extracellular domain with adissociation constant of less than or equal to about 10⁻⁷ M.
 6. Themethod of claim 1, wherein the monoclonal antibody or antigen bindingfragment thereof binds to the same epitope as the antibody produced bythe hybridoma having ATCC Designation No. PTA-10663.
 7. The method ofclaim 1, wherein the monoclonal antibody or antigen binding fragmentthereof inhibits the binding of both Wnt1 and Wnt3a to LRP6.
 8. Themethod of claim 1, further comprising the step of administering a secondagent to the subject, wherein the second agent has a synergistic effectwith the monoclonal antibody.
 9. The method of claim 8, wherein thesecond agent is selected from the group consisting of an anti-angiogenicagent, an anti-VEGF reagent, and combinations thereof.
 10. The method ofclaim 1, wherein the condition is fibrosis.
 11. The method of claim 1,wherein the condition is colon cancer.
 12. A method of inhibiting and/ordecreasing the occurrence and/or severity of at least one conditionselected from the group consisting of inflammation, vascular leakage,fibrosis, abnormal neovascularization, and cancer, said methodcomprising the step of: administering to a subject suffering from orpredisposed to the at least one condition a pharmaceutical composition,wherein the pharmaceutical composition inhibits activation of the Wntsignaling pathway, thereby inhibiting and/or decreasing the occurrenceand/or severity of the at least one condition, and wherein thepharmaceutical composition comprises a monoclonal antibody and apharmaceutically acceptable carrier, and wherein the monoclonal antibodyis produced by the hybridoma having ATCC Designation No. PTA-10663. 13.The method of claim 12, further comprising the step of administering asecond agent to the subject, wherein the second agent has a synergisticeffect with the monoclonal antibody.
 14. The method of claim 13, whereinthe second agent is selected from the group consisting of ananti-angiogenic agent, an anti-VEGF reagent, and combinations thereof.15. The method of claim 12, wherein the condition is fibrosis.
 16. Themethod of claim 12, wherein the condition is colon cancer.
 17. A methodof inhibiting and/or decreasing the occurrence and/or severity of atleast one condition selected from the group consisting of inflammation,vascular leakage, fibrosis, abnormal neovascularization, and cancer,said method comprising the step of: administering to a subject sufferingfrom or predisposed to the at least one condition a pharmaceuticalcomposition, wherein the pharmaceutical composition inhibits activationof the Wnt signaling pathway, thereby inhibiting and/or decreasing theoccurrence and/or severity of the at least one condition, and whereinthe pharmaceutical composition comprises a monoclonal antibody orantigen binding fragment thereof and a pharmaceutically acceptablecarrier, wherein the monoclonal antibody or antigen binding fragmentthereof specifically binds to the LRP6 extracellular domain having theamino acid sequence of SEQ ID NO: 2, and wherein the isolated monoclonalantibody or antigen binding fragment thereof comprises a heavy chainvariable region and a light chain variable region, and wherein the heavychain variable region has the amino acid sequence of SEQ ID NO:5 and/orthe light chain variable region has the amino acid sequence of SEQ IDNO:7.
 18. The method of claim 17, wherein the monoclonal antibody orantigen binding fragment thereof is a single domain antibody.
 19. Themethod of claim 17, further comprising the step of administering asecond agent to the subject, wherein the second agent has a synergisticeffect with the monoclonal antibody.
 20. The method of claim 19, whereinthe second agent is selected from the group consisting of ananti-angiogenic agent, an anti-VEGF reagent, and combinations thereof.21. The method of claim 17, wherein the condition is fibrosis.
 22. Themethod of claim 17, wherein the condition is colon cancer.